| Histoplasma capsulatum is a dimorphic pathogenic fungus. In the soil, H. capsulatum grows in a mycelial form. When soil containing H. capsulatum is disturbed, mycelial fragments or conidia (vegetative-spores) become aerosolized. Once inhaled by a mammalian host, mycelial fragments and/or conidia convert into a yeast-like form. H. capsulatum yeast escape innate immune defenses and colonize host macrophages during infection. After the onset of adaptive immunity, activated macrophages produce the antimicrobial effector nitric oxide ( •NO) to restrict H. capsulatum replication. However, despite exposure to reactive nitrogen species (RNS), H. capsulatum is able to establish persistent infections. To understand how H. capsulatum copes with RNS-induced damage, we used a shotgun genomic microarray to examine the transcriptional response of H. capsulatum to •NO-generating compounds. We identified 695 microarray clones that were induced greater than 4-fold upon nitrosative stress. Because our microarray clones were generated from random fragments of genomic DNA, the induced clones did not necessarily correlate with H. capsulatum genes. Instead, the induced clones represented regions of interest that likely overlapped portions of RNS-induced genes. To identify the RNS-induced transcripts, we created tiling microarrays in which both strands of the regions of interest were represented by short oligonucleotides tiled side-by-side. Using gene expression analysis, we determined the genomic boundaries and coding strand of 153 RNS-induced transcripts. Homologs of these genes in other organisms are involved in iron acquisition, energy production, protein folding/degradation, DNA repair, and •NO detoxification. One of these genes, a P450 nitric oxide reductase homolog we named NOR1, was characterized in detail. Ectopic expression of NOR1 was sufficient to confer increased resistance to RNS in culture. We propose that H. capsulatum uses the pathways identified here to cope with RNS-induced damage during pathogenesis. |
