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

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 (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, , was important for fungal survival . Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the response, we infected mice with a reporter yeast strain and assessed expression at 0, 3, 7, and 14 days post-infection (dpi). expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased expression. This increase was dependent on another cytokine that activates MΦ to control replication, M-CSF. These findings illustrate the reporter's ability to sense Zn and and correlate expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of yeasts, one of which is the limitation of trace metals. On the other hand, combats metal restriction by upregulating metal importers such as the Zn importer . This transporter contributes to pathogenesis upon activation of adaptive immunity. We constructed a fluorescent transcriptional reporter to probe Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host.