A monoclonal antibody to Histoplasma capsulatum alters the intracellular fate of the fungus in murine macrophages

Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.     

Utilization and cell-surface binding of hemin by Histoplasma capsulatum

Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection. Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages. However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection. Thus the ability of H. capsulatum to use hemin and heme-containing compounds was examined. Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture. The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator. Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron. The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface. The ability of H. capsulatum to bind hemin was related to the nutritional status of the cells. Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator. Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin. Additionally, the pre-incubation of iron-starved H. capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin. These results suggest that H. capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.     

Knocking on the right door and making a comfortable home: Histoplasma capsulatum intracellular pathogenesis

Histoplasma capsulatum is a successful intracellular pathogen of mammalian macrophages. As such, this fungus must survive and/or subvert hostile environmental onslaughts in a professionally antimicrobial host cell. H. capsulatum uses different host receptors for binding to macrophages (beta 2 integrins) than it uses for binding to dendritic cells (the fibronectin receptor); the fungus experiences different degrees of success in survival in these two cells. Surface expression of HSP60 as the specific adhesin for macrophage beta 2 integrins represents a novel mechanism for binding. Long considered a resident of the phagolysosome, H. capsulatum may also reside in a modified phagosome without experiencing phagolysosomal fusion. H. capsulatum must compete with the host to acquire the essential nutrient iron, and has several potential mechanisms for accomplishing this necessary feat. Finally, H. capsulatum displays morphotype-specific expression of several genes, and a calcium-binding protein expressed only by the pathogenic yeast phase has been demonstrated as essential for full virulence. An organism's environment is of great importance to its success or failure, and H. capsulatum is good at finding or making the right environment in the host.     

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains' attenuated virulence in mice and macrophages.     

Complex requirements for nascent and memory immunity in pulmonary histoplasmosis.

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive alpha/beta TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-gamma production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-alpha, but not IFN-gamma, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-alpha. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-alpha.     

Comparative study of a new Chrysosporium species with Histoplasma capsulatum

The Chrysosporium state of a new ascomycete, Renispora flavissima (Gymnoascaceae), resembles Histoplasma capsulatum in its macroconidial morphology. It was discovered growing in bat guano, from which it was readily isolated by direct plating of diluted soil, but only rarely from mice inoculated with soil suspensions. The fungus was consistently reisolated from tissues of mice inoculated intravenously and intraperitoneally with conidial and mycelial suspensions from cultures of the fungus. Nevertheless, there is no evidence that this species is pathogenic. Cultures grew at 37 degrees C, but did not convert to a yeast form on agar media or within cultured mouse peritoneal macrophages. Although the hyphae and conidia of this fungus fluoresce when stained with H. capsulatum fluorescent antibodies, exoantigens of the fungus produce neither H nor M precipitin bands, thus differentiating it from H. capsulatum. Both H. capsulatum and the new Chrysosporium sp. demonstrate isozyme polymorphism, and isozymic differences have been discussed between the two species.     

Efficacy of cell-free antigens in evaluating cell immunity and inducing protection in a murine model of histoplasmosis

Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when mycelial fragments are inhaled. Resistance to H. capsulatum is dependent on cellular immunity mediated by T cells and macrophages. Here we standardized the production of extracts containing cell-free antigens (CFAgs) and observed their efficacy in evaluating cellular immunity during murine histoplasmosis. CFAgs induced a more potent delayed-type hypersensitivity (DTH) response in H. capsulatum-infected mice than did histoplasmin-a classical antigen. This DTH response to CFAgs is able to determine the immune status of infected mice and to predict their death. Moreover, CFAgs stimulated spleen cells from immune mice to produce higher amounts of gamma interferon (IFN-gamma) in vitro. Finally, immunization with CFAgs protected against a lethal inoculum of H. capsulatum. These results demonstrate that CFAgs may be useful for the evaluation of cellular immune response and as a potential source for the development of a vaccine against histoplasmosis.     

Comparative study of the effect of thiabendazole and fungizone on Histoplasma capsulatum in macrophages.

The antifungal properties of Fungizone (amphotericin B intravenous solution) and thiabendazole on Histoplasma capsulatum within guinea pig macrophages were compared using the staining method and a newly developed plating method to determine the viability of intracellular H. capsulatum. The two methods were compared to determine the effectiveness of Fungizone and thiabendazole on H. capsulatum within macrophages. Fungizone was fungicidal for intracellular H. capsulatum, killing 99.9% of the yeasts at a concentration of 0.5 microgram/ml. There was some indication that non-viable intracellular yeasts were stained which could result in misinterpretation of the effectiveness of Fungizone using the staining method unless the yeasts are very closely examined for staining abnormalities. There was a good correlation between the two methods to demonstrate suppression of the multiplication of intracellular H. capsulatum in macrophages treated with 50 microgram/ml of thiabendazole. Thiabendazole was lethal for some intracellular H. capsulatum.     

Effect of Temperature on the Intracellular Growth of Histoplasma capsulatum

The rate and form of growth of Histoplasma capsulatum within histiocytes derived from homothermic and poikilothermic animals, and incubated at 25, 30, and 37 C, are described. The generation time of the fungus in mouse cells incubated at 37 and 25 C was 11 and 24 hr, respectively. Blastospore formation was progressively retarded in cells at 25 C, and this retardation was accompanied by germination of some of the blastospores. The generation time of the fungus in mouse cells incubated at 30 C was the same as it was at 37 C. Germ tube formation was not a prominent feature of intracellular growth at 30 C. The rate of growth of H. capsulatum within frog histiocytes at 30 and 25 C was slower than it was in mouse cells at the same temperatures. Some loss of frog histiocytes in cultures incubated at 37 C prevented accurate estimation of the rate of growth of the fungus at this temperature. Growth of H. capsulatum in frog histiocytes kept at 25 C was progressively retarded, and the retardation was accompanied by germination of the yeasts. Yeast-phase growth predominated in fish histiocytes incubated at 30 C, whereas germ tubes were formed within such cells incubated at 25 C. Cell survival of fish histiocytes was relatively poor in culture, and no estimates of rate of growth of the fungus within these cells were made.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Histoplasma capsulatum yeasts are phagocytosed via very late antigen-5, killed, and processed for antigen presentation by human dendritic cells

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As the induction of cell-mediated immunity to Hc is of critical importance in host defense, we sought to determine whether dendritic cells (DC) could function as a primary APC for this pathogenic fungus. DC obtained by culture of human monocytes in the presence of GM-CSF and IL-4 phagocytosed Hc yeasts in a time-dependent manner. Upon ingestion, the intracellular growth of yeasts within DC was completely inhibited compared with rapid growth within human macrophages. Electron microscopy of DC with ingested Hc revealed that many of the yeasts were degraded as early as 2 h postingestion. In contrast to macrophages, human DC recognized Hc yeasts via the fibronectin receptor, very late Ag-5, and not via CD18 receptors. DC stimulated Hc-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of viable and heat-killed Hc yeasts, but greater proliferation was achieved after ingestion of viable yeasts. These data demonstrate that human DC can phagocytose and degrade a fungal pathogen and subsequently process the appropriate Ags for stimulation of lymphocyte proliferation. In vivo, such interactions between DC and Hc may facilitate the induction of cell-mediated immunity.     

Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment

Histoplasma capsulatum is a thermally dimorphic ascomycete that is a significant cause of respiratory and systemic disease in mammals including humans, especially immunocompromised individuals such as AIDS patients. As an environmental mold found in the soil, it is a successful member of a competitive polymicrobial ecosystem. Its host-adapted yeast form is a facultative intracellular pathogen of mammalian macrophages. H. capsulatum faces a variety of environmental changes during the course of infection and must survive under harsh conditions or modulate its microenvironment to achieve success as a pathogen. Histoplasmosis may be considered the fungal homolog of the bacterial infection tuberculosis, since both H. capsulatum and Mycobacterium tuberculosis exploit the macrophage as a host cell and can cause acute or persistent pulmonary and disseminated infection and reactivation disease. The identification and functional analysis of biologically or pathogenically important H. capsulatum genes have been greatly facilitated by the development of molecular genetic experimental capabilities in this organism. This review focuses on responsiveness of this fungus to its environment, including differential expression of genes and adaptive phenotypic traits.     

An alpha-(1,4)-amylase is essential for alpha-(1,3)-glucan production and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic fungus that causes respiratory and systemic disease and is capable of surviving and replicating within macrophages. The virulence of Histoplasma has been linked to cell wall alpha-(1,3)-glucan; however, the role of this polysaccharide during infection, its organization within the cell wall, and its synthesis and regulation remain poorly understood. To identify genes involved in the biosynthesis of alpha-(1,3)-glucan, we employed a forward genetics strategy to isolate physically marked mutants with reduced alpha-(1,3)-glucan. Insertional mutants were generated in a virulent strain of H. capsulatum by optimization of Agrobacterium tumefaciens-mediated transformation. Approximately 90% of these mutants possessed single insertions with no chromosomal rearrangements or deletions in the host genome. To confirm the role and specificity of identified candidate genes, we phenocopied the disrupted locus by either RNA interference or targeted gene deletion. Our findings indicate alpha-(1,3)-glucan production requires the function of the AMY1 gene product, a novel protein with homology to the alpha-amylase family of glycosyl hydrolases, and UGP1, a UTP-glucose-1-phosphate uridylyltransferase which synthesizes UDP-glucose monomers. Loss of AMY1 function attenuated the ability of Histoplasma to kill macrophages and to colonize murine lungs.     

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP–GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.     

Generation of IL-23 producing dendritic cells (DCs) by airborne fungi regulates fungal pathogenicity via the induction of T(H)-17 responses

Interleukin-17 (IL-17) producing T helper cells (T(H)-17) comprise a newly recognized T cell subset with an emerging role in adaptive immunity to a variety of fungi. Whether different airborne fungi trigger a common signaling pathway for T(H)-17 induction, and whether this ability is related to the inherent pathogenic behavior of each fungus is currently unknown. Here we show that, as opposed to primary pathogenic fungi (Histoplasma capsulatum), opportunistic fungal pathogens (Aspergillus and Rhizopus) trigger a common innate sensing pathway in human dendritic cells (DCs) that results in robust production of IL-23 and drives T(H)-17 responses. This response requires activation of dectin-1 by the fungal cell wall polysaccharide b-glucan that is selectively exposed during the invasive growth of opportunistic fungi. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma not only abrogates the pathogenicity of this fungus, but also triggers the induction of IL-23 producing DCs. Thus, b-glucan exposure in the fungal cell wall is essential for the induction of IL-23/T(H)-17 axis and may represent a key factor that regulates protective immunity to opportunistic but not pathogenic fungi.     

Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum

Histoplasmosis, due to the intracellular fungus Histoplasma capsulatum, can be diagnosed by demonstrating the presence of antibodies specific to the immunodominant M antigen. However, the role of this protein in the pathogenesis of histoplasmosis has not been elucidated. We sought to structurally and immunologically characterize the protein, determine yeast cell surface expression, and confirm catalase activity. A 3D-rendering of the M antigen by homology modeling revealed that the structures and domains closely resemble characterized fungal catalases. We generated monoclonal antibodies (mAbs) to the protein and determined that the M antigen is present on the yeast cell surface and in cell wall/cell membrane preparations. Similarly, we found that the majority of catalase activity was in extracts containing fungal surface antigens and that the M antigen is not significantly secreted by live yeast cells. The mAbs also identified unique epitopes on the M antigen. The localization of the M antigen to the cell surface of H. capsulatum yeast and the characterization of the protein's major epitopes have important implications since it demonstrates that although the protein may participate in protecting the fungus against oxidative stress it is also accessible to host immune cells and antibody.     

Macrophage-independent fungicidal action of the pulmonary collectins

Histoplasma capsulatum (Hc) is a facultative intracellular fungal pathogen that causes acute and chronic pneumonia. In this study, we investigated the role of the pulmonary collectins, surfactant proteins (SP) A and D, in the clearance of Hc yeast from the lung. Exposure of yeast to either collectin induced a dose-dependent decrease in [3H]leucine incorporation by several strains of Hc. This decrement was attributed to killing of the collectin-exposed yeast since it failed to grow on agar medium. Exposure to SP-A or -D resulted in increased yeast permeability based on a leak of protein from the organism and enhanced access of an impermeant substrate to intracellular alkaline phosphatase. Inbred and outbred SP-A null (-/-) mice were modestly more susceptible to pulmonary infection with Hc than strain and age-matched SP-A (+/+) control mice. The increase in susceptibility was associated with a decrement in the number of CD8+ cells in the lungs of SP-A-/- mice. Neither SP-A nor SP-D inhibited the growth of macrophage-internalized Hc. We conclude that the SP-A and SP-D are antimicrobial proteins that directly inhibit the growth of Hc by increasing permeability of the organism and that Hc gains asylum from collectin-mediated killing by rapid entry into pulmonary macrophages.     

Cysteine transport and sulfite reductase activity in a germination-defective mutant of Histoplasma capsulatum.

A mutant of the dimorphic, zoopathogenic fungus Histoplasma capsulatum, defective in the ability of its blastospores to germinate, was used to show that the nutritional requirement for cysteine and the expression of sulfite reductase activity are temperature dependent and not related to the growth form the fungus, whereas the kinetics of cysteine transport depend on both temperature and the form of the fungus.