Yeast Transcriptome and In Vivo Hypoxia Detection Reveals Histoplasma capsulatum Response to Low Oxygen Tension

Although there is growing understanding of the microenvironmental conditions fungal pathogens encounter as they colonize their host, nothing is known about Histoplasma capsulatum's response to hypoxia. Here we characterized hypoxia during murine histoplasmosis using an in vivo hypoxia detection agent, Hypoxyprobe-2 (HP-2); and analyzed H. capsulatum's transcriptional profile in response to in vitro hypoxia. Immunohistopathology and flow cytometry analyses revealed distinct regions of hypoxia during infection. Granuloma cells, enriched with macrophages and T-cells isolated from infected livers were 66-76% positive for HP-2, of which, 95% of macrophages and 55% of T-cells were hypoxic. Although inhibited, H. capsulatum was able to survive under in vitro hypoxic conditions (<1% O2), and restored growth when replaced in normoxia. Next-generation sequencing (RNA-seq) analysis after 24 hours of hypoxia demonstrated a significant increase in NIT50 (swirm domain DNA binding protein), a predicted ABC transporter (ABC), NADPH oxidoreductase (NADP/FAD), and guanine nucleotide exchange factor (RSP/GEF); and other genes with no known designated function. Computational transcription factor binding site analysis predicted human sterol regulatory element binding protein (SREBP) binding sites upstream of NIT50, ABC, NADP/FAD and RSP/GEF. Hypoxia resulted in a time-dependent increase in the H. capsulatum homolog of SREBP, here named Srb1. Srb1 peaked at 8 hours and returned to basal levels by 24 hours. Our findings demonstrate that H. capsulatum encounters and survives severe hypoxia during infection. Additionally, the hypoxic response may be regulated at the level of transcription, and these studies contribute to the understanding of hypoxic regulation and adaptation in H. capsulatum.