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 (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, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 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 ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the 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 transcriptional reporter to probe H. capsulatum 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 in vivo 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.

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 (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte-MΦ 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-γ, stimulated MΦ. 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 MΦ and exudate MΦ. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 activity with GM-CSF and M-CSF activation of MΦ. Importance: Phagocytes 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.

Incorporation of the HIV-1 envelope glycoprotein into viral particles is regulated by the tubular recycling endosome in a cell type-specific manner.

The HIV-1 envelope glycoprotein (Env) is incorporated into particles during assembly on the plasma membrane (PM). Env initially reaches the PM through the secretory pathway, after which it is rapidly endocytosed via an AP-2- and clathrin-dependent mechanism. Here we show that endocytosed cell surface Env enters the tubular recycling endosome compartment (TRE). Trafficking to the TRE was dependent upon motifs within the CT previously implicated in Env recycling and particle incorporation. Depletion of TRE components MICAL-L1 or EHD1 led to defects in Env incorporation, particle infectivity, and viral replication. Remarkably, defects were limited to cell types defined as nonpermissive for incorporation of CT-deleted Env, including monocyte-derived macrophages, and not observed in 293T, HeLa, or MT-4 cells. This work identifies the TRE as an essential component of Env trafficking and particle incorporation, and provides evidence that the cell type-dependent incorporation of Env is defined by interactions with components of the TRE.

A metabolic inhibitor arms macrophages to kill intracellular fungal pathogens by manipulating zinc homeostasis.

Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients, including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource-poor environment by marshaling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog 2-deoxy-D-glucose (2-DG) armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages, decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This effect promoted the antifungal machinery of macrophages and expanded the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.

Dual-purpose isocyanides produced by Aspergillus fumigatus contribute to cellular copper sufficiency and exhibit antimicrobial activity.

The maintenance of sufficient but nontoxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that, in Aspergillus fumigatus, xanthocillin and other isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay, and inductively coupled plasma mass spectrometry analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with a variety of human pathogenic fungi and bacteria established copper-dependent antimicrobial properties of xan BGC metabolites, including inhibition of laccase activity. Remediation of xanthocillin-treated Pseudomonas aeruginosa growth by copper supported the copper-chelating properties of xan BGC isocyanide products. The existence of the xan BGC in several filamentous fungi suggests a heretofore unknown role of eukaryotic natural products in copper homeostasis and mediation of interactions with competing microbes.

Metallothionein 3 Controls the Phenotype and Metabolic Programming of Alternatively Activated Macrophages.

Alternatively activated (M2) macrophages promote wound healing but weaken antimicrobial defenses. The mechanisms that enforce macrophage divergence and dictate the phenotypic and metabolic characteristics of M2 macrophages remain elusive. We show that alternative activation with interleukin (IL)-4 induces expression of metallothionein 3 (MT3) that regulates macrophage polarization and function. MT3 was requisite for metabolic reprograming in IL-4-stimulated macrophages or M(IL-4) macrophages to promote mitochondrial respiration and suppress glycolysis. MT3 fostered an M(IL-4) phenotype, suppressed hypoxia inducible factor (HIF)1α activation, and thwarted the emergence of a proinflammatory M1 program in macrophages. MT3 deficiency augmented macrophage plasticity, resulting in enhanced interferon γ (IFNγ) responsiveness and a dampened M(IL-4) phenotype. Thus, MT3 programs the phenotype and metabolic fate of M(IL-4) macrophages.