Utilization of a Histoplasma capsulatum zinc reporter reveals the complexities of fungal sensing of metal deprivation

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (M{Phi}) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. M{Phi} activated by granulocyte-M{Phi} colony stimulating factor (GM-CSF) induce metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the high affinity zinc importer, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under the control of this importer. This reporter accurately responds to medium devoid of Zn. ZRT2 expression increased ([~]5-fold) in GM-CSF, but not interferon-{gamma}, stimulated M{Phi}. To examine the in vivo response, we infected mice with reporter yeasts and assessed ZRT2 expression at 0-, 3-, 7-, and 14-days post-infection (dpi). ZRT2 expression minimally increased at 3-dpi and peaked on 7-dpi, corresponding with onset of adaptive immunity. We discovered that the major phagocyte populations that restrict Zn to the fungus are interstitial M{Phi} and exudate M{Phi}. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates M{Phi} to control H. capsulatum replication, M-CSF. These findings illustrate the reporters ability to sense Zn in vitro and in vivo and correlate ZRT2 activity with GM-CSF and M-CSF activation of M{Phi}. ImportancePhagocytes use an arsenal of defenses to control replication of Histoplasma yeasts, one of which is limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probe H. capsulatum Zn sensing during infection and exposed a role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to studying how intracellular pathogens sense and respond to the changing environments of the host.