| The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. However, little is known about the fungal genes required for intracellular replication in the host. The ability to scavenge iron from the host is important to virulence, and yet, too much iron is toxic to the cell. Thus, iron acquisition is a highly regulated process. In response to excess iron, H. capsulatum represses expression of genes involved in iron uptake via a GATA transcription factor, SRE1. The molecular mechanism by which Sre1p negatively regulates gene expression was studied through the use of various biochemical and molecular techniques. Expression of SRE1 was reduced under iron-starving conditions, underscoring its role as a negative regulator of genes involved in iron uptake. DNA binding assays indicated that Sre1p specifically bound DNA containing the 5'-(G/A)ATC(T/A)GATAA-3' sequence, which is found in the promoters of iron-regulated genes.;The mode by which Sre1p binds DNA was investigated by examining its metal content. Metal analyses indicated that a substoichiometric amount of iron (∼0.6 equiv), predominately Fe3+, was bound to the purified protein. In addition to zinc, iron was necessary for full DNA-binding activity of Sre1p. Furthermore, mutations of key cysteine residues in a conserved region of Sre1p, led to a decrease in bound iron. The loss of iron led to a ∼2.5-fold decrease in DNA-binding affinity, indicating that iron was directly involved in SRE1 regulation of iron uptake genes. These results were incorporated into a model for how SRE1 regulates iron acquisition in H. capsulatum.;In addition to studying the mechanism of iron regulation, the potential role of a putative cytochrome P450 nitric oxide (NO) reductase, No1p, in detoxifying NO, an antimicrobial effector produced by macrophages, was also investigated. Similar to iron, high concentrations of NO may cause oxidative damage to the cell. Kinetic and spectral studies of No1p, revealed that it could metabolize NO by reducing it to the less toxic gas, N2O. The reaction rate was similar to other previously characterized NO metabolizing enzymes that have been implicated in NO detoxification during infection. |
