Identification ofHistoplasma capsulatumTranscripts Induced in Response to Reactive Nitrogen Species

Abstract

The pathogenic fungus Histoplasma capsulatum escapes innate immune defenses and colonizes host macrophages during infection. After the onset of adaptive immunity, the production of the antimicrobial effector nitric oxide ( ⅐ NO) restricts H. capsulatum replication. However, H. capsulatum can establish persistent infections, indicating that it survives in the host despite exposure to reactive nitrogen species (RNS). To understand how H. capsulatum responds to RNS, we determined the transcriptional profile of H. capsulatum to ⅐ NO-generating compounds using a shotgun genomic microarray. We identified 695 microarray clones that were induced >4-fold upon nitrosative stress. Because our microarray clones were generated from random fragments of genomic DNA, they did not necessarily correspond to H. capsulatum open reading frames. To identify induced genes, we used high-density oligonucleotide tiling arrays to determine the genomic boundaries and coding strand of 153 RNS-induced transcripts. Homologues of these genes in other organisms are involved in iron acquisition, energy production, stress response, protein folding/degradation, DNA repair, and ⅐ NO detoxification. Ectopic expression of one of these genes, a P450 nitric oxide reductase homologue, was sufficient to increase resistance of H. capsulatum to RNS in culture. We propose that H. capsulatum uses the pathways identified here to cope with RNS-induced damage during pathogenesis.

Fungal denitrification and nitric oxide reductase cytochrome P450nor

“…P450nor is therefore a multi-functional detoxifying enzyme. P450nor is also related to the pathogenicity of a fungus [48].…”

Heme-Biosynthetic Porphobilinogen Deaminase Protects Aspergillus nidulans from Nitrosative Stress

“…No functions for PBG-D in the RNS response have been reported on the basis of the transcriptomes of S. cerevisiae, C. albicans, and H. capsulatum (6,14,17,25). This might be due to the relatively lower induction level of hemC transcription by RNS than of the genes characterized by the transcriptome analyses.…”

“…The synthesized protoheme serves as a cofactor for FHb and enables RNS tolerance, which implies that hemC responds to RNS and regulates protoheme synthesis for adaptation to nitrosative stress conditions. Reports have shown that RNS also induce the expression of genes for some fungal hemoproteins, including catalases and peroxidases, which facilitate tolerance of the oxidative stress caused by RNS (22,25). The A. nidulans isozymes respond to and facilitate the tolerance of oxidative stress (13,18), although their role in the RNS response remains obscure.…”

“…S-Nitrosoglutathione (GSNO) reductase, which is conserved from bacteria to humans, reduces GSNO to ammonia and glutathione disulfide and consequently enhances cellular resistance to nitrosative stress (20). The genome of Aspergillus nidulans encodes FHb and GSNO reductase orthologs, and no other RNSdetoxifying enzyme has been identified in the fungus.Transcriptome analyses have shown that more than 100 genes of Saccharomyces cerevisiae, Candida albicans, and Histoplasma capsulatum fungi are regulated by NO (14,17,25). The functions of a large number of these genes are unknown, indicating that the fungal mechanisms of the RNS response are not fully understood.…”

“…Transcriptome analyses have shown that more than 100 genes of Saccharomyces cerevisiae, Candida albicans, and Histoplasma capsulatum fungi are regulated by NO (14,17,25). The functions of a large number of these genes are unknown, indicating that the fungal mechanisms of the RNS response are not fully understood.…”