Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

Restraint of Fumarate Accrual by HIF-1α Preserves miR-27a-Mediated Limitation of Interleukin 10 during Infection of Macrophages by Histoplasma capsulatum.

Hypoxia-inducible factor 1α (HIF-1α) regulates the immunometabolic phenotype of macrophages, including the orchestration of inflammatory and antimicrobial processes. Macrophages deficient in HIF-1α produce excessive quantities of the anti-inflammatory cytokine interleukin 10 (IL-10) during infection with the intracellular fungal pathogen Histoplasma capsulatum (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565-579, 2016, https://doi.org/10.4049/jimmunol.1600342). Thus, the macrophage fails to become activated in response to proinflammatory cytokines and remains the intracellular niche of the pathogen. Here, we identify the tricarboxylic acid (TCA) cycle metabolite fumarate as the driver of IL-10 during macrophage infection with H. capsulatum in the absence of HIF-1α. Accumulation of fumarate reduced expression of a HIF-1α-dependent microRNA (miRNA), miR-27a, known to mediate decay of Il10 mRNA. Inhibition of fumarate accrual in vivo limited IL-10 and fungal growth. Our data demonstrate the critical role of HIF-1α in shaping appropriate TCA cycle activity in response to infection and highlight the consequences of a dysregulated immunometabolic response. IMPORTANCE Histoplasma capsulatum and related Histoplasma species are intracellular fungal pathogens endemic to broad regions of the globe, including the Americas, Africa, and Asia. While most infections resolve with mild or no symptoms, failure of the host to control fungal growth produces severe disease. Previously, we reported that loss of a key transcriptional regulator, hypoxia-inducible factor 1α (HIF-1α), in macrophages led to a lethal failure to control growth of Histoplasma (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565-579, 2016, https://doi.org/10.4049/jimmunol.1600342). Inhibition of phagocyte activation due to excessive interleukin 10 by HIF-1α-deficient macrophages drove this outcome. In this study, we demonstrate that HIF-1α maintains contextually appropriate TCA cycle metabolism within Histoplasma-infected macrophages. The absence of HIF-1α results in excessive fumarate production that alters miRNA-27a regulation of interleukin-10. HIF-1α thus preserves the capacity of macrophages to transition from a permissive intracellular niche to the site of pathogen killing.

Pulmonary Mycosis Drives Forkhead Box Protein A2 Degradation and Mucus Hypersecretion through Activation of the Spleen Tyrosine Kinase-Epidermal Growth Factor Receptor-AKT/Extracellular Signal-Regulated Kinase 1/2 Signaling.

Pulmonary mycoses are difficult to treat and detrimental to patients. Fungal infections modulate the lung immune response, induce goblet cell hyperplasia and metaplasia, and mucus hypersecretion in the airways. Excessive mucus clogs small airways and reduces pulmonary function by decreasing oxygen exchange, leading to respiratory distress. The forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. However, little is known whether pulmonary mycosis modulates FOXA2 function. Herein, we investigated whether Blastomyces dermatitidis and Histoplasma capsulatum-infected canine and feline lungs and airway epithelial cells could serve as higher animal models to examine the relationships between fungal pneumonia and FOXA2-regulated airway mucus homeostasis. The results indicate that fungal infection down-regulated FOXA2 expression in airway epithelial cells, with concomitant overexpression of mucin 5AC (MUC5AC) and mucin 5B (MUC5B) mucins. Mechanistic studies reveal that B. dermatitidis infection, as well as ß-glucan exposure, activated the Dectin-1-SYK-epidermal growth factor receptor-AKT/extracellular signal-regulated kinase 1/2 signaling pathway that inhibits the expression of FOXA2, resulting in overexpression of MUC5AC and MUC5B in canine airway cells. Further understanding of the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics against excessive mucus in both human and veterinary patients with pulmonary mycosis.

Clinical outcomes and cortical reserve in adrenal histoplasmosis-A retrospective follow-up study of 40 patients.

OBJECTIVE: Detailed studies of Addison's disease resulting from disseminated adrenal histoplasmosis (AH) are not available. We describe the presentation and prognosis of AH and cortisol status before and after antifungal therapy. DESIGN: Single-centre retrospective hospital-based study of 40 consecutive adults with AH [39 males; age (mean ± SD) 53 ± 11 years] was conducted between 2006 and 2018. The median duration of follow-up was 2.5 years (range 0.2-12 years). PATIENTS AND METHODS: AH was diagnosed by bilateral adrenal enlargement on CT scan and presence of Histoplasma by histology and/or culture of biopsied adrenal tissue. All patients received oral itraconazole and, if required, amphotericin B as per guidelines. ACTH-stimulated serum cortisol (normal > 500 nmol/L) was measured in 38 patients at diagnosis and re-tested after one year of antifungal therapy in 21 patients. RESULTS: Seventy-three per cent of patients had primary adrenal insufficiency (PAI) and one-third had an adrenal crisis at presentation. HIV antibody was negative in all patients. Of the 29 patients who completed antifungal therapy, 25 (86%) were in remission at last follow-up. Overall, 8 (20%) patients died: three had a sudden death, four had severe histoplasmosis and one died due to adrenal crisis. No patient with PAI became eucortisolemic on re-testing after one year of antifungal therapy. Of the eight patients with normal cortisol at diagnosis, two developed adrenal insufficiency on follow-up. CONCLUSION: All patients with AH tested negative for HIV antibody. While patients achieved a high rate of clinical remission after antifungal therapy, overall mortality was significant. Cortisol insufficiency did not normalize despite treatment.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages.

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus' EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host's immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

Tumor Necrosis Factor Alpha Antagonism Reveals a Gut/Lung Axis That Amplifies Regulatory T Cells in a Pulmonary Fungal Infection.

Tumor necrosis factor (TNF) antagonists are popular therapies for inflammatory diseases. These agents enhance the numbers and function of regulatory T cells (Tregs), which are important in controlling inflammatory diseases. However, elevated Treg levels increase susceptibility to infections, including histoplasmosis. We determined the mechanism by which Tregs expand in TNF-neutralized mice infected with Histoplasma capsulatum Lung CD11c+ CD11b+ dendritic cells (DCs), but not alveolar macrophages, from H. capsulatum-infected mice treated with anti-TNF induced a higher percentage of Tregs than control DCs in vitro CD11b+ CD103+ DCs, understood to be unique to the intestines, were augmented in lungs with anti-TNF treatment. In the absence of this subset, DCs from anti-TNF-treated mice failed to amplify Tregs in vitro CD11b+ CD103+ DCs from TNF-neutralized mice displayed higher retinaldehyde dehydrogenase 2 (RALDH2) gene expression, and CD11b+ CD103+ RALDH+ DCs exhibited greater enzyme activity. To determine if CD11b+ CD103+ DCs migrated from gut to lung, fluorescent beads were delivered to the gut via oral gavage, and the lungs were assessed for bead-containing DCs. Anti-TNF induced migration of CD11b+ CD103+ DCs from the gut to the lung that enhanced the generation of Tregs in H. capsulatum-infected mice. Therefore, TNF neutralization promotes susceptibility to pulmonary H. capsulatum infection by promoting a gut/lung migration of DCs that enhances Tregs.

Differentiation of the fungus Histoplasma capsulatum into a pathogen of phagocytes.

Mammalian body temperature triggers differentiation of the fungal pathogen Histoplasma capsulatum into yeast cells. The Drk1 regulatory kinase and an interdependent network of Ryp transcription factors establish the yeast state. Beyond morphology, the differentiation-dependent expression program equips yeasts for invasion and survival within phagosomes. Yeast cells produce α-glucan and the Eng1 endoglucanase which hide yeasts from immune detection. Secretion of yeast phase-specific Sod3 and CatB detoxify phagocyte-derived reactive oxygen molecules. Histoplasma cells adapt to iron and zinc limitation in activated macrophages by production of siderophores and the Zrt2 transporter, respectively. Yeasts also respond to inflammation-associated hypoxia. Histoplasma pathogenicity thus relies on factors controlled by yeast differentiation as well as environment-dependent responses.

Protective Effect of Galectin-1 during Histoplasma capsulatum Infection Is Associated with Prostaglandin E2 and Nitric Oxide Modulation.

Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1-/- mice exhibited elevated levels of prostaglandin E2 (PGE2, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE2 as an important lipid mediator in the pathogenesis of histoplasmosis.

IL-4 Induces Metallothionein 3- and SLC30A4-Dependent Increase in Intracellular Zn(2+) that Promotes Pathogen Persistence in Macrophages.

Alternative activation of macrophages promotes wound healing but weakens antimicrobial defenses against intracellular pathogens. The mechanisms that suppress macrophage function to create a favorable environment for pathogen growth remain elusive. We show that interleukin (IL)-4 triggers a metallothionein 3 (MT3)- and Zn exporter SLC30A4-dependent increase in the labile Zn(2+) stores in macrophages and that intracellular pathogens can exploit this increase in Zn to survive. IL-4 regulates this pathway by shuttling extracellular Zn into macrophages and by activating cathepsins that act on MT3 to release bound Zn. We show that IL-4 can modulate Zn homeostasis in both human monocytes and mice. In vivo, MT3 can repress macrophage function in an M2-polarizing environment to promote pathogen persistence. Thus, MT3 and SLC30A4 dictate the size of the labile Zn(2+) pool and promote the survival of a prototypical intracellular pathogen in M2 macrophages.

The Eng1 ß-Glucanase Enhances Histoplasma Virulence by Reducing ß-Glucan Exposure.

UNLABELLED: The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes ß-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall ß-glucans, which results in enhanced binding to the Dectin-1 ß-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed ß-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize ß-glucan exposure: α-glucan provides a masking function by covering the ß-glucan-rich cell wall, while Eng1 removes any remaining exposed ß-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed ß-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes. IMPORTANCE: The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a ß-glucanase, Eng1, which reduces recognition of fungal cell wall ß-glucans. We demonstrated that the Eng1 ß-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed ß-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host ß-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall ß-glucans by α-glucan and enzymatic removal of exposed ß-glucans by the Eng1 ß-glucanase.

Cutaneous presentation of disseminated histoplasmosis as a solitary peri-anal ulcer. Case report and discussion.

Disseminated histoplasmosis has a diverse and non-specific range of clinical signs and symptoms. In a significant minority of patients, cutaneous lesions are apparent at the time of initial presentation, affording an opportunity to establish the diagnosis from a skin biopsy. The most frequently reported clinical scenario in immunocompromised patients with cutaneous involvement is that of multiple papulo-nodular lesions on the face, trunk or extremities. The following report features an immunocompetent patient who presented with a solitary ulcerated plaque on the buttocks close to the anal verge. This case presentation underscores the broad spectrum of clinical presentations as well as the potential for diagnostic confusion with protozoa such as Leishmania or Trypanosoma species during histopathologic examination if special stains for fungal organisms are not performed.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.

The leukotriene B4/BLT1 axis is a key determinant in susceptibility and resistance to histoplasmosis.

The bioactive lipid mediator leukotriene B4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype.

Histoplasma capsulatum depends on de novo vitamin biosynthesis for intraphagosomal proliferation.

During infection of the mammalian host, Histoplasma capsulatum yeasts survive and reside within macrophages of the immune system. Whereas some intracellular pathogens escape into the host cytosol, Histoplasma yeasts remain within the macrophage phagosome. This intracellular Histoplasma-containing compartment imposes nutritional challenges for yeast growth and replication. We identified and annotated vitamin synthesis pathways encoded in the Histoplasma genome and confirmed by growth in minimal medium that Histoplasma yeasts can synthesize all essential vitamins with the exception of thiamine. Riboflavin, pantothenate, and biotin auxotrophs of Histoplasma were generated to probe whether these vitamins are available to intracellular yeasts. Disruption of the RIB2 gene (riboflavin biosynthesis) prevented growth and proliferation of yeasts in macrophages and severely attenuated Histoplasma virulence in a murine model of respiratory histoplasmosis. Rib2-deficient yeasts were not cleared from lung tissue but persisted, consistent with functional survival mechanisms but inability to replicate in vivo. In addition, depletion of Pan6 (pantothenate biosynthesis) but not Bio2 function (biotin synthesis) also impaired Histoplasma virulence. These results indicate that the Histoplasma-containing phagosome is limiting for riboflavin and pantothenate and that Histoplasma virulence requires de novo synthesis of these cofactor precursors. Since mammalian hosts do not rely on vitamin synthesis but instead acquire essential vitamins through diet, vitamin synthesis pathways represent druggable targets for therapeutics.

An element of life: competition for zinc in host-pathogen interaction.

Zinc homeostasis plays a plethora of different roles in the immune response. In this issue of Immunity, Vignesh et al. (2013) demonstrate that stimulation of macrophages with GM-CSF deprives intracellular Histoplasma capsulatum of zinc, improving pathogen clearance.