Date of Graduation
5-2014
Document Type
Dissertation (PhD)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Michael C. Lorenz, Ph.D.
Committee Member
Kevin A. Morano, Ph.D.
Committee Member
Ambro van Hoof, Ph.D.
Committee Member
Hung Ton-That, Ph.D.
Committee Member
Jeffrey A. Frost, Ph.D
Abstract
Candida albicans is the most importan thuman-associatedfungus.It is a commensal microorganism but also an opportunistic pathogen able to cause superficial infections aswellaslife-threateninginfectionswhich are associated with a highmortalityrateof 50%. The interactions between C. albicans and the cells of the mammalian innate immune system, which confer the most important protecting mechanisms against disseminated infections, are very dynamic and determine the success of C. albicans as a pathogen. Transcriptional profiling has shown that phagocytosis of C. albicans by macrophages results primarily in the activation of alternative carbon metabolism pathways suggesting that the pathogen is exposed to a glucose poor environment. Changes in amino acid metabolism are not observed, with the only exception of arginine biosynthesis. The strong upregulation of the arginine biosynthetic (ARG) genes suggests their importancefor C. albicans virulence.
Using single cell reporters I have shown that the (ARG) genes are induced specifically upon C. albicans phagocytosis in response to ROS produced by the murine macrophages. I have also shown that these genes are not regulated by the general amino acid control response, and that Gcn4, the master regulator of this response in S. cerevisiae, is a negative regulator of the ARG genes in C. albicans. C. albicans also has homologs for some components of the S. cerevisiae ArgR/Mcm1 negative regulatory complex. Using promoter dissection and computational analyses, I have identified putative binding sites for some of these regulators in the promoter of the ARG genes that function in response to arginine and ROS. Using site-directed mutagenesis and single cell reporters I have shown that there are discrete positive and negative cis acting elements consistent with the promoter dissection studies that regulate the expression of the ARG genes in response to arginine and phagocytosis.
Together these studies have shown that the ARG genes in C. albicans are induced upon phagocytosis by murine macrophages due to the exposure of the fungal cells to ROS. In addition, the regulation of these genes by arginine limitation and ROS exposure overlaps, but it is not yet known how ROS induces expression of these genes.
Keywords
Candida albicans, macrophages, arginine, amino acids, phagocytosis