Date of Award

Summer 7-2014

Embargo Period

2-23-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Aaron P. Mitchell

Second Advisor

Frederick Lanni

Abstract

The fungal pathogen Candida albicans is a leading cause of device-associated and other nosocomial infections. The traits of biofilm formation and invasion into an underlying surface are important for Candida to cause disease. In this dissertation, I describe my work, which reveals a novel role for glycerol in C. albicans biofilm formation and hyphal invasion. Through genomewide expression profiling it was observed that glycerol biosynthetic genes were highly upregulated in biofilms relative to the planktonic (suspension) cultures. Consistent with this observation, cells in a biofilm also accumulated higher amounts of glycerol then non-biofilm cells. In order to study the impact of glycerol on biofilm formation I made a deletion mutant, rhr2Δ/Δ, in the gene encoding glycerol-3-phosphatase. Under in vitro conditions, the rhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. The mutant is also severely defective in biofilm formation in the rat venous catheter model of biofilm infection. Surprisingly, genome-wide expression profiling showed that the rhr2Δ/Δ mutant has reduced expression of the cell-surface adhesin genes: ALS1, ALS3, and HWP1, as well as many other genes that are up regulated in biofilms. The role of Rhr2 in adherence and biofilm formation depends on adhesin gene expression as overexpression of any of the adhesin genes restores biofilm formation by rhr2Δ/Δ in vitro and in vivo. Thus, our findings indicate that glycerol plays a regulatory role in biofilm gene expression and that the adhesin genes are among the functional Rhr2-regulated genes. I observed that the functional significance of biofilm glycerol accumulation lies in its ability to generate turgor to drive hyphal invasion. I showed that using an assay for invasive growth into elastic polyacrylamide. Using mathematical and biophysical approaches, I show that C. albicans can generate turgor equivalent to 20 atm in order to invade. Additionally, I show that several mutants with deletions in biofilm and hyphal regulator genes are defective in invasion, thus 4 implying a critical role for biofilm formation and hyphal morphogenesis in invasion. The glyceroldeficient hyphae, however, cannot invade even when they are hyperadherent and enmeshed in a confluent biofilm. Thus, my observations suggest that Candida hyphae can generate high turgor while in a biofilm which is required for invasion into elastic substrata. I predict that turgor is required for hyphal invasion of mucosal surfaces as well, and that is significant for the pathogenicity of this fungus. Among the other biofilm up-regulated genes, I focused my attention on SHB17 that encodes the enzyme sedoheptulose bisphosphatase, involved in nonoxidative arm of ribose biosynthesis. I made deletion mutant strains and analyzed the mutants for their abilities to form biofilm. I observed the mutant to have a moderate loss of biofilm biomass. The mutant biofilm, however, contained hyphae that were more slender in appearance than either the wild type or the complemented strain. I additionally observed that the mutant phenotype is biofilm specific and ribose-dependent, as the mutant biofilms grown in ribose-containing growth medium do not display the slender hyphal phenotype. Thus, these observations suggest that biofilm growth drives higher flux through the nonoxidative arm of ribose synthesis. Any alterations to this flux can affect the cell morphology. In the course of Rhr2 studies, I developed an improved method for biofilm imaging. I applied this approach to analyze the roles of the adhesin Als1 and biofilm regulators Bcr1 and Brg1 in biofilm formation. These studies helped to establish that Als1 stimulates Brg1 activity in Bcr1-dpendent manner.

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