Candida albicans is a common opportunistic fungal pathogen that causes serious infections in immunocompromised patients, and discomforting mucosal and cutaneous infections in healthy individuals. Like many pathogenic bacteria, the ability of C. albicans to acquire iron from the infected host is required for virulence. To obtain this essential metal C. albicans uses three biochemically distinct high-affinity uptake systems. A reductive system, requiring CaFTR1, CFL95, and CaFET99 is able to reduce and release iron from transferrin. A siderophore system mediates uptake of the ferrichrome type siderophores and requires the transporter encoded by CaSIT1. Finally, a heme iron utilization system requires heme oxygenase encoded by CaHMX1. Although these iron uptake systems consist of unique protein components, they are coordinately up-regulated by iron starvation. This suggests that a common iron regulatory system exists. The goal of this project is to identify the cis-acting and trans-acting elements involved in the iron regulation of these uptake pathways.
In Aim 1 we will identify the cis-acting iron response elements that we propose are present in these iron regulated genes. This will be achieved by promoter deletion analysis with lacZ reporter constructs. These in vitro experiments will be guided in part by computer analysis of the iron regulated promoters.
In Aim 2 we will test the hypothesis that CaAFT1 encodes the iron regulatory factor. CaAFT1 is homologous to the transcriptional iron regulator AFT1 of Saccharomyces cerevisiae. A conditional mutant of CaAFT1 will be constructed and the iron acquisition genes will be examined for loss of regulation. The CaAftl protein will be tested for the ability to bind DNA regions identified in Aim 1, and in vivo genomic footprinting will be performed. If no evidence is obtained that CaAFT1 is involved in iron regulation we will initiate the search for the iron regulatory factor. Extracts prepared from iron starved and iron replete C. albicans cells will be examined for their ability to bind DNA elements identified in Aim1.
|Knight, Simon A B; Dancis, Andrew (2006) Reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) is dependent on CaFRE10 ferric reductase for Candida albicans grown in unbuffered media. Microbiology 152:2301-8|