The investigation of the histidine biosynthetic pathway for drug target validation in the pathogenic yeast Candida albicans
Abstract
The goal of this study was to increase the understanding of how the histidine biosynthetic genes are regulated in Candida albicans, and to determine if the biosynthesis of histidine is required for the pathogenicity of C. albicans. The CaHIS6 and CaHIS7 genes were cloned from C. albicans and shown to encode a 5′-ProFAR isomerase and an IGP synthase, the enzymes that catalyze the fourth and fifth steps of the histidine biosynthetic pathway, respectively. Bacterial complementation and standardized enzymatic assays were used to show that CaHIS6 and CaHIS7 genes encode functional histidine biosynthetic proteins. To determine if the histidine biosynthetic genes in C. albicans were regulated by a general control mechanism, histidine starvation was chemically induced by using a weak inhibitor (3-aminotriazole) and a high affinity inhibitor (IRL1803) of the HIS3 protein. The expression of CaHIS3, CaHIS6, and CaHIS7 genes were analyzed under these growth conditions to determine if they were under GCN4 global control. Interestingly, the CaHIS3 and CaHIS7 genes, but not the CaHIS6 gene, were shown to be regulated by GCN4. The concentration of IRL1803 that inhibited the growth of a C. albicans wild-type strain by 50% was determined to be 10 μg/mL. This is the first report of an inhibitor of a histidine biosynthetic enzyme having a fungistatic effect in C. albicans. A cahis7 knockout strain was produced by using the URA-blaster strategy. Using this cahis7 deficient strain, in vitro growth experiments determined the amount of histidine required for growth and survival. Additionally, interaction of the knockout strain with host cells was studied using a mouse macrophage model. The ability of the knockout strain to evade the macrophage response was compared to that of a wild-type strain. Both strains were engulfed by the macrophages and evaded the macrophage killing response in a similar time dependent manner. These regulatory and knockout studies lay the groundwork for future studies that will probe the relevance of the histidine biosynthetic enzymes as targets for inhibitor-based antifungal drug design.
Degree
Ph.D.
Advisors
Davisson, Purdue University.
Subject Area
Microbiology|Molecular biology
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