Deletion of the Stress Response Gene DDR48 from Histoplasma capsulatum Increases Sensitivity to Oxidative Stress, Increases Susceptibility to Antifungals, and Decreases Fitness in Macrophages

Blancett, Logan T.; Runge, Kauri A.; Reyes, Gabriella M.; Kennedy, Lauren; Jackson, Sydney C.; Scheuermann, Sarah E.; Harmon, Mallory B.; Williams, Jamease C.; Shearer, Glenmore

doi:10.3390/jof7110981

Cited by 4 publications

(3 citation statements)

References 86 publications

(115 reference statements)

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“…H. capsulatum RNA was isolated by mechanical disruption with 0.5 mm glass beads, extracted with TRIzol (Invitrogen), and precipitated with ethanol, as previously described ( 49 , 50 ). Genomic DNA was removed via DNase digestion and column purification using the PureLink RNA Mini Kit (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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

Blancett,

Evans,

Candor

et al. 2024

mSphere

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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. IMPORTANCE Phagocytes 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.

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Section: Methodsmentioning

confidence: 99%

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

Blancett,

Evans,

Candor

et al. 2024

mSphere

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“…H. capsulatum RNA was isolated by mechanical disruption with 0.5 mm glass beads, extraction with TRIzol™ (Invitrogen), and precipitated with ethanol, as previously described (44, 45). Genomic DNA was removed via DNase digestion and column purification using the PureLink™ RNA Mini Kit (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Utilization of aHistoplasma capsulatumzinc reporter reveals the complexities of fungal sensing of metal deprivation

Blancett,

Evans,

Candor

et al. 2023

Preprint

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Histoplasma capsulatumis 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 controlH. capsulatumreplication, M-CSF. These findings illustrate the reporter’s ability to sense Znin vitroandin vivoand correlate ZRT2 activity with GM-CSF and M-CSF activation of MΦ.ImportancePhagocytes use an arsenal of defenses to control replication ofHistoplasmayeasts, one of which is limitation of trace metals. On the other hand,H. capsulatumcombats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes toH. capsulatumpathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probeH. capsulatumZn 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.

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“…While fungal QS research is still in its infancy, its discovery has transformed our view of the fungal kingdom and may eventually contribute to the advancement of new antifungal therapies. The fungi known to possess QS include Candida albicans [9], Saccharomyces cerevisiae [10], Histoplasma capsulatum [11], Cryptococcus neoformans [12], Neurospora crassa [13], Ceratocystis ulmi , [14] and Ophiostoma floccosum [15]. Meanwhile, research on QS regulation in Aspergillus , Penicillium , and other filamentous fungi has made significant breakthroughs.…”

Section: Introductionmentioning

confidence: 99%

The regulatory functions of oxylipins in fungi: A review

et al. 2023

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Quorum sensing (QS) is a communication mechanism between microorganisms originally found in bacteria. In recent years, an important QS mechanism has been discovered in the field of fungi, namely, the lipoxygenase compound oxylipin of arachidonic acid acts as a QS molecule in life cycle control, particularly in the sexual and asexual development of fungi. However, the role of oxylipins in mediating eukaryotic communication has not been previously described. In this paper, we review the regulatory role of oxylipins and the underlying mechanisms and discuss the potential for application in major fungi. The role of oxylipin as a fungal quorum‐sensing molecule is the main focus of the review. Besides, the quorum regulation of fungal morphological transformation, biofilm formation, virulence factors, secondary metabolism, infection, symbiosis, and other physiological behaviors are discussed. Moreover, future prospectives and applications are elaborated as well.

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Deletion of the Stress Response Gene DDR48 from Histoplasma capsulatum Increases Sensitivity to Oxidative Stress, Increases Susceptibility to Antifungals, and Decreases Fitness in Macrophages

Cited by 4 publications

References 86 publications

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

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

Utilization of aHistoplasma capsulatumzinc reporter reveals the complexities of fungal sensing of metal deprivation

The regulatory functions of oxylipins in fungi: A review

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