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Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development. IMPORTANCE It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence. In this context, fungal lipid metabolism is an excellent candidate for developing drugs due to its essential roles in cellular scaffolds, energy storage, and signaling transductors. Here, we provide a detailed map of Histoplasma capsulatum lipid metabolism. The map revealed points of this fungus lipid metabolism that can be targeted for developing antifungal drugs.

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum , must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo , indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages. IMPORTANCE Histoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment., (Copyright © 2020 Shen et al.)

Background: Disseminated Kaposi sarcoma (DKS) is present in patients with advanced HIV infection in whom co-infection with other opportunistic pathogens can occur. Bone marrow (BM) aspirate and biopsy comprise a robust diagnostic tool in patients with fever, cytopenias, and abnormal liver tests. However, the yield in patients with DKS has not been determined., Objective: The aim of this study was to evaluate the utility of BM aspirate and biopsy in patients with DKS., Methods: We included 40 male patients with a recent diagnosis of DKS. BM aspirate and biopsy was performed as part of the workup to rule out co-infections., Results: In four patients, Mycobacterium avium complex (MAC) was recovered from culture. In other four patients, intracellular yeasts were observed in the Grocott stain, diagnosed as Histoplasma. The yield of BM was calculated in 20%. Only 12 patients (30%) had fever and 11 (27.5%) had pancytopenia. Alkaline phosphatase (ALP) above normal values and C-reactive protein (CRP) were higher in patients with positive results for BM than in those with negative results (63% vs. 21.9%, and 3.0 vs. 1.2 mg/L; p = 0.03 in both comparisons). No differences were found when complete blood-count abnormalities were compared., Conclusion: We recommend performing a BM aspirate for stains, culture, and biopsy in all HIV patients with DKS, as this will permit the early diagnosis of co-infections and prevent further complications in those who receive chemotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America., (Copyright © 2020. Published by Elsevier España, S.L.U.)

The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.

Epidemiological cut-off values (ECVs) have been used as a tool to detect the acquisition of resistance mechanisms to antifungal drugs. In this context, the objective of this study was to determine the ECVs for classic antifungals against Histoplasma capsulatum var. capsulatum isolates from human immunodeficiency virus (HIV)-infected patients with a diagnosis of disseminated histoplasmosis. First, minimum inhibitory concentrations (MICs) for amphotericin B (AmB), itraconazole (ITR), fluconazole (FLU), voriconazole (VCZ) and caspofungin (CAS) were determined against 138 H. capsulatum isolates in the filamentous form by the broth microdilution method; antifungal ECVs were then calculated. MIC ranges were 0.0078-1 µg/mL for AmB, 0.0005-0.0625 µg/mL for ITR, 2 to ≥256 µg/mL for FLU, 0.0078-1 µg/mL for VCZ and ≤0.0156 to ≥32 µg/mL for CAS. The obtained ECVs were 0.5, 0.0313, 128, 0.5 and 16 µg/mL for AmB, ITR, FLU, VCZ and CAS, respectively. The percentage of wild-type isolates was 96.4% for AmB, 98.6% for ITR and 99.3% for FLU, VCZ and CAS. Although these results do not cover all phylogenetic species of H. capsulatum, they bring important information on strains from Brazil. In addition, the assessed isolates were from HIV-positive patients, which may not reflect the antifungal ECVs against isolates from immunocompetent individuals or from other sources. Finally, this study pioneers the initiative of establishing ECVs for five antifungal agents against H. capsulatum var. capsulatum, providing a criterion for the interpretation of susceptibility results as well as a monitoring strategy for the emergence of antifungal resistance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Histoplasma capsulatum (Hc) is the causative agent for the respiratory infection histoplasmosis. The fungus exists in the environment as a saprophytic multi-cellular mould. Spores are inhaled by mammals whereupon the organism will convert into the single-celled yeast morphotype resulting in infection. The shift to the yeast morphotype is required for pathogenesis. Most studies on dimorphism have examined yeast-phase-specific genes and few mould-phase-specific genes have been investigated. It is likely, that some mould-phase-specific genes must be downregulated for the yeast to form or upregulated for the mould to form. We isolated a strongly expressed mould-specific gene, M46, from an expression library enriched for mould upregulated genes in Hc strain G186AS. To determine if M46 is involved in dimorphism, M46 was ectopically expressed in yeast phase growing temperature, and an m46 knockout strain was created via allelic replacement. Ectopically expressing M46 in yeast, did not induce filamentous growth. Genomic disruption of M46 by allelic replacement did not alter the morphology of the mould as seen in bright field microscopy, scanning electron microscopy, and transmission electron microscopy. A growth curve study, revealed that M46 is not involved in maintaining the growth rate of cells. These findings indicate that the mould specific M46 gene is not necessary nor essential for dimorphism, maintaining the normal mould morphology, and growth rate of Histoplasma capsulatum., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis., (© 2015 John Wiley & Sons Ltd.)

Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aims: This study aimed to evaluate the in vitro activity of miltefosine and levamisole against strains of Coccidioides posadasii in the filamentous phase and strains of Histoplasma capsulatum in filamentous and yeast phases., Methods and Results: Strains of C. posadasii in the filamentous phase (n = 22) and strains of H. capsulatum in filamentous (n = 40) and yeast phases (n = 13) were, respectively, submitted to broth macrodilution and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of miltefosine and levamisole. The effect of the drugs on cell membrane permeability under osmotic stress conditions and total ergosterol production were also assessed, along with quantification of extravasated molecules. The results show the inhibitory effect of levamisole and miltefosine against C. posadasii and H. capsulatum and the effect of these drugs on ergosterol synthesis and the permeability of the plasma membrane using subinhibitory concentrations against strains subjected to osmotic stress. Levamisole was also able to cause the release of nucleic acids., Conclusions: Miltefosine and levamisole are capable of inhibiting the in vitro growth of C. posadasii and H. capsulatum, probably by altering the permeability of the cellular membrane., Significance and Impact of the Study: This work presents alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of the use of miltefosine and levamisole as adjuvants in antifungal therapy, providing perspectives for the design of in vivo studies., (© 2015 The Society for Applied Microbiology.)

The host pulmonary response to the fungus Histoplasma capsulatum was evaluated, through the profile of cytokines detected by the MagPix magnetic beads platform in lung homogenates and by lung-granulomas formation, from mice intra-nasally infected with mycelial propagules (M-phase) of two virulent H. capsulatum strains, EH-46 and G-217B. Results highlight that mice lung inflammatory response depends on the H. capsulatum strain used, during the first step of the fungal infection. IL-1β and TNF-α increased their concentrations in mice infected with both strains. The highest levels of IL-6, IL-17, and IL-23 were found in EH-46-infected mice, whereas levels of IL-22 were variable at all post-infection times for both strains. Significant increases of IL-12, IFN-γ, IL-4, and IL-10 were associated to EH-46-infected mice. Histological lung findings from EH-46-infected mice revealed incipient and numerous well-developed granulomas, distributed in lung-lobes at the 14th and the 21st days after infection, according to cytokine profiles., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B, and farnesol alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (fractional inhibitory concentration index ≤0.5) between itraconazole and farnesol was found against 100 and 83.3 % of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5 and 44.4 % of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs, itraconazole and amphotericin B, and farnesol alone, and the combination of itraconazole and farnesol, were tested against mature biofilms of H. capsulatum, through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) metabolic assay, and the itraconazole and amphotericin B showed lower antibiofilm activity when compared to farnesol alone and farnesol combined with itraconazole. In conclusion, farnesol showed promising results as an antifungal agent against H. capsulatum and also showed adjuvant action, especially when combined with itraconazole, increasing the fungal susceptibility to this drug., (© 2015 The Authors.)

The present paper is an overview of the primary events that are associated with the histoplasmosis immune response in the murine model. Valuable data that have been recorded in the scientific literature have contributed to an improved understanding of the clinical course of this systemic mycosis, which is caused by the dimorphic fungus Histoplasma capsulatum. Data must be analyzed carefully, given that misinterpretation could be generated because most of the available information is based on experimental host-parasite interactions that used inappropriate proceedings, i.e., the non-natural route of infection with the parasitic and virulent fungal yeast-phase, which is not the usual infective phase of the etiological agent of this mycosis. Thus, due to their versatility, complexity, and similarities with humans, several murine models have played a fundamental role in exploring the host-parasite interaction during H. capsulatum infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus (HIV)-associated disseminated Histoplasma capsulatum capsulatum infection often mimics tuberculosis. This disease is well know in the United States but is dramatically underdiagnosed in Central and South America. In the Amazon region, given the available incidence data and the regional HIV prevalence, it is expected that, every year, 1,500 cases of histoplasmosis affect HIV patients in that region alone. Given the mortality in undiagnosed patients, at least 600 patients would be expected to die from an undiagnosed but treatable disease. The lack of a simple diagnostic tool and the lack of awareness by clinicians spiral in a vicious cycle and made a major problem invisible for 30 years. The HIV/acquired immunodeficiency syndrome community should tackle this problem now to prevent numerous avoidable deaths from HIV-associated histoplasmosis in the region and elsewhere.

Paracoccidioides brasiliensis and P. lutzii are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis (PCM). Previously, we characterized the PbMDJ1 gene. This gene encodes P. brasiliensis chaperone Mdj1, which in yeast is a mitochondrial member of the J-domain family, whose main function is to regulate cognate Hsp70 activities. We produced rabbit polyclonal antibody antirecombinant PbMdj1 (rPbMdj1), which labeled the protein not only in mitochondria but also at the cell wall of P. brasiliensis yeasts of isolate Pb18. Here we used anti-rPbMdj1 in confocal microscopy to localize Mdj1 in Pb18 and other fungal isolates grown at different temperatures. Dual intracellular and cell surface pattern were initially seen in yeast-phase P. brasiliensis Pb3, Pb18 (control), P. lutzii Pb01, and Histoplasma capsulatum. Pb18 and Aspergillus fumigatus hyphae as well as Pb3 pseudo hyphae formed at 36°C were labeled predominantly along the cell surface. Preferential surface localization was observed by 72 h of yeast-mycelium thermotransition. It was interesting to observe that anti-rPbMdj1 concentrated at the surface tip and branching points of A. fumigatus hyphae grown at 36°C, suggesting a role in growth, whereas at 23°C, anti-rPbMdj1 was distributed along the hyphal surface. In Pb3, Pb18, and Pb01 mitochondrial extracts, the antibodies revealed a specific 55-kDa band, which corresponds to the processed Mdj1 size. The presence of Mdj1 on the fungal cell wall suggests that this protein could also play a role in the interaction with the host.

As eukaryotes, fungi possess relatively few molecules sufficiently unique from mammalian cell components to be used as drug targets. Consequently, most current antifungals have significant host cell toxicity. Primary fungal pathogens (e.g., Histoplasma) are of particular concern, as few antifungals are effective in treating them. To identify additional antifungal candidates for the treatment of histoplasmosis, we developed a high-throughput platform for monitoring Histoplasma growth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibited Histoplasma yeast growth were identified. Compound 41F5 has fungistatic activity against Histoplasma yeast at micromolar concentrations, with a 50% inhibitory concentration (IC50) of 0.87 μM, and has the greatest selectivity for yeast (at least 62-fold) relative to host cells. Structurally, 41F5 consists of an aminothiazole core with an alicyclic substituent at the 2-position and an aromatic substituent at the 5-position. 41F5 inhibits Histoplasma growth in liquid culture and similarly inhibits yeast cells within macrophages, the actual host environment of this fungal pathogen during infection. Importantly, 41F5 protects infected host cells from Histoplasma-induced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal for Histoplasma infections.

This study evaluated the in vitro interaction between ciprofloxacin (CIP) and classical antifungals against Histoplasma capsulatum var. capsulatum in mycelial (n = 16) and yeast-like forms (n = 9) and Coccidioides posadasii in mycelial form (n = 16). This research was conducted through broth microdilution and macrodilution, according to Clinical Laboratory Standards Institute. Inocula were prepared to obtain from 0.5 × 10(3) to 2.5 × 10(4)  cfu ml(-1) for H. capsulatum and from 10(3) to 5 × 10(3)  cfu ml(-1) for C. posadasii. Initially, minimum inhibitory concentration (MIC) for each drug alone was determined. Then, these MICs were used as the highest concentration for each drug during combination assays. The procedures were performed in duplicate. For all combination assays, MICs were defined as the lowest concentration capable of inhibiting 80% of visible fungal growth, when compared to the drug-free control. Drug interaction was evaluated by paired sample t-Student test. The obtained data showed a significant MIC reduction for most tested combinations of CIP with antifungals, except for that of CIP and voriconazole against yeast-like H. capsulatum. This study brings potential alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of using CIP as an adjuvant antifungal therapy, providing perspectives to delineate in vivo studies., (© 2012 Blackwell Verlag GmbH.)

Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma., (Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I-Hc II) and a region of approximately 600-bp (ITS1-ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100 % sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I-Hc II in the presence of the ITS1-ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100 %. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.

The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Published by Elsevier Inc.)

Regulation of iron acquisition genes is critical for microbial survival under both iron-limiting conditions (to acquire essential iron) and iron-replete conditions (to limit iron toxicity). In fungi, iron acquisition genes are repressed under iron-replete conditions by a conserved GATA transcriptional regulator. Here we investigate the role of this transcription factor, Sre1, in the cellular responses of the fungal pathogen Histoplasma capsulatum to iron. We showed that cells in which SRE1 levels were diminished by RNA interference were unable to repress siderophore biosynthesis and utilization genes in the presence of abundant iron and thus produced siderophores even under iron-replete conditions. Mutation of a GATA-containing consensus site found in the promoters of these genes also resulted in inappropriate gene expression under iron-replete conditions. Microarray analysis comparing control and SRE1-depleted strains under conditions of iron limitation or abundance revealed both iron-responsive genes and Sre1-dependent genes, which comprised distinct but overlapping sets. Iron-responsive genes included those encoding putative oxidoreductases, metabolic and mitochondrial enzymes, superoxide dismutase, and nitrosative-stress-response genes; Sre1-dependent genes were of diverse functions. Genes regulated by iron levels and Sre1 included all of the siderophore biosynthesis genes, a gene involved in reductive iron acquisition, an iron-responsive transcription factor, and two catalases. Based on transcriptional profiling and phenotypic analyses, we conclude that Sre1 plays a critical role in the regulation of both traditional iron-responsive genes and iron-independent pathways such as regulation of cell morphology. These data highlight the evolving realization that the effect of Sre1 orthologs on fungal biology extends beyond the iron regulon.

Infections due to Histoplasma capsulatum occur as a result of the inhalation of airborne microconidia of the mold into the alveoli of the lungs. In this study we quantified the transformation over time of conidia into yeast-like cells within macrophages (MΦ) and dendritic cells (DC). Conidia from strain G217B which had been surface labeled with carboxy-fluorescein succinimidyl ester (CFSE), or conidia from strain G217B that expresses green fluorescent protein (GFP) only in the yeast phase, were used to infect MΦ and DC. At various time points, numbers of intracellular conidia or yeasts were quantified via phase-contrast and fluorescent microscopy. Transformation of conidia from non-GFP-expressing G217B also was quantified by their incorporation of ³H-leucine. In both human and murine MΦ, numerous yeast-like cells appeared by day 3 post-infection. The time course of conidia transformation into yeasts in culture medium was the same as in MΦ. However, transformation of conidia to yeasts was significantly restricted in human DC and murine lung DC. In DC, significant numbers of yeasts did not appear until 5 days post-infection. Further, MΦ monolayers were destroyed by day 6-7 post-infection, whereas DC monolayers remained intact throughout the study period. These data suggest that in vivo, conidia may transform into yeast-like cells efficiently whether or not they are phagocytosed by MΦ, but not when ingested by DC.

Histoplasma capsulatum strains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype II Histoplasma virulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucan in vitro, yet they remain fully virulent in vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishes AGS1 expression. Nonetheless, AGS1 mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced during in vivo growth despite its absence in vitro. To directly test whether AGS1 contributes to chemotype I strain virulence, we prevented AGS1 function by RNA interference and by insertional mutation. Loss of AGS1 function in chemotype I does not impair the cytotoxicity of ags1(-) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, the ags1(-) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate that AGS1 expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a unique Histoplasma chemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

We report a case of Histoplasma capsulatum endocarditis in which Histoplasma antigen assay and fungal blood cultures were negative. The diagnosis was made by microscopic examination and culture of the excised valve. Histoplasma capsulatum should be considered in the differential diagnosis of culture-negative endocarditis in regions where it is endemic and in travelers.

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented > or =98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.

The human fungal pathogen Histoplasma capsulatum grows in a sporulating filamentous form in the soil and, after inhalation of infectious spores, converts to a pathogenic yeast form inside host macrophages in response to temperature. Here we report the identification of two genes (RYP2 and RYP3) required for yeast-phase growth. Ryp2 and Ryp3 are homologous to each other and to the Velvet A family of regulatory proteins in Aspergillus species and other filamentous fungi. Wild-type H. capsulatum grows as filaments at room temperature and as yeast cells at 37 degrees C, but ryp2 and ryp3 mutants constitutively grow as filaments independent of temperature. RYP2 and RYP3 transcripts accumulate to higher levels at 37 degrees C than at room temperature. This differential expression is similar to the previously identified RYP1 transcript, which encodes a transcriptional regulator required for the yeast-phase expression program. Ryp1 associates with the upstream region of RYP2, and each of the three RYP genes is required for the differential expression of the others at 37 degrees C. In addition to responding to the elevated temperature of the mammalian host, RYP2 and RYP3 are essential for viable spore production and regulation of sporulation at room temperature. This regulatory function is strikingly similar to the role of the Aspergillus Velvet A protein family in spore development in response to light, with the notable distinction that the H. capsulatum circuit responds to temperature.

Therapeutic effects of Sodium Iodide (NaI), Potassium Iodide (KI), ground berries of "Endod" (Phytolacca dodecandra) and Penstrip were evaluated on 70 cases of equine hitoplasmosis (EH). Response to each treatment was assessed using clinical examination of the lesions. Statistically significant difference (P = 0.0036) in therapeutic effect was observed among the different remedies. Cases treated either with a combination of NaI and Penstrip (F = 6.34, P = 0.004) or "Endod" and Penstrip (F = 3.64, P = 0.031) demonstrated significant response. The difference in response to treatment between early and advanced cases of EH was statistically significant (t = 2.22, P = 0.0148).

The dimorphism of Histoplasma reflects a developmental switch in morphology and lifestyle that is necessary for virulence. The dimorphism regulating kinase DRK1 and the Histoplasma WOR1 homolog RYP1 mediate the thermally induced transition to the pathogenic yeast-phase program. The genes expressed as part of this regulon influence the host-pathogen interaction to favor Histoplasma virulence. While surface localized HSP60 supports yeast attachment to host macrophages, yeast alpha-glucan polysaccharides conceal immunostimulatory cell wall beta-glucans from detection by macrophage receptors. Intramacrophage growth of yeast cells is facilitated by CBP a secreted, protease-resistant calcium-binding protein tailored to function within the phagolysosomal environment. In some Histoplasma strains, YPS3 promotes dissemination of yeast from pulmonary infection sites. The Histoplasma yeast-phase program includes additional cell surface and extracellular molecules that potentially function in further aspects of Histoplasma virulence.

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.

Histoplasma capsulatum, a fungal pathogen of humans, switches from a filamentous spore-forming mold in the soil to a pathogenic budding-yeast form in the human host. This morphologic switch, which is exhibited by H. capsulatum and a group of evolutionarily related fungal pathogens, is regulated by temperature. Using insertional mutagenesis, we identified a gene, RYP1 (required for yeast phase growth), which is required for yeast-form growth at 37 degrees C. ryp1 mutants are constitutively filamentous irrespective of temperature. Ryp1 is a member of a family of fungal proteins that includes Wor1, a master transcriptional regulator of the white-opaque transition required for mating in Candida albicans. Ryp1 associates with its own upstream regulatory region, consistent with a direct role in transcriptional control, and both the protein and its transcript accumulate to high levels in wild-type yeast-phase cells. Microarray analysis demonstrated that Ryp1 is required for the expression of the vast majority of yeast-specific genes, including two genes linked to virulence. Thus, Ryp1 appears to be a critical transcriptional regulator of a temperature-regulated morphologic switch in H. capsulatum.

Lipids contribute significantly to the pathogenesis of fungal infectious diseases and an understanding of lipid metabolism occurring in fungal pathogens can help the development of more efficient antifungal therapeutic strategies. In this study, the effect of culture age on the distribution of fatty acids among different neutral lipid (NL) classes in the dimorphic fungus Histoplasma capsulatum was investigated. Yeast cells of the G217B strain grown in two different media were collected after 4 and 7 days of growth, which roughly correspond to log and stationary culture growth phases, respectively. Neither culture age nor medium type had any influence on qualitative fatty acid (FA) profiles; however, the FA percentage composition varied with culture growth. A culture age-related decrease in the content of unsaturated FAs could be observed in all four of the NL classes examined, but the most intensive changes were detected in diacylglycerol and free FA fractions. Conversely, an increase in saturated FAs was observed. The transcriptional analysis of two major delta 9- and delta 12-FA desaturase genes, ode1 and sde1, showed no differences in their expression levels under experimental conditions. These results showing the dynamics of changes in FA composition in the NL fraction were concomitant with nutrient exhaustion in aging H. capsulatum cultures. Overall, the results presented in this work not only have implications for our knowledge of basic lipid biochemistry of H. capsulatum, but also will contribute to better understanding of biology and pathogenesis of this fungus and, consequently, can help in the discovery of more effective antifungal drugs.

The pathogenic fungus Histoplasma capsulatum causes disease ranging from mild to fatal in healthy and immunocompromised humans. Infection rates reach 80% in endemic areas, including the Midwestern United States. We used inbred mice to identify a 300-fold difference in fungal burden. A/J mice showed lower fungal burden and morbidity than C57BL/6J mice, a reversal of the trend observed for many bacterial pathogens. We mapped the differences in fungal burden to discrete locations on chromosomes 1, 6, 15 and 17 with high significance. Substitution of a single resistant chromosome 17 onto the susceptible background was sufficient to lower fungal burden. These loci will allow dissection of the fungal-specific immune program.

Epizootic lymphangitis (EL) was experimentally reproduced in four horses that had been purchased from an EL-free district. Two horses were injected with either 0.2 mL of the yeast form of Histoplasma capsulatum var. farciminosum (HCF) in pus (Horse 1), or 0.2 mL (ca. 20 mg) of a suspension in saline of the mycelial form (Horse 2), both into the pre-scapular and pre-femoral lymph nodes, with scarification of the skin of the left hind limb, conjunctiva of the right eye and the nasal membrane of the right nostril. The two other horses served as controls. Nodular lesions of EL appeared during the fourth week of infection at all sites in the horse infected with the yeast form. Lesions only appeared in the lymph nodes and skin scratches of the horse infected with the mycelial suspension after three months. The control horses showed no clinical signs. The yeast form was recovered from the lesions of both infected horses. Similarly, the mycelial form was isolated from both horses on Sabouraud's Dextrose Agar. This experiment showed the reproducibility of clinical EL through experimental infection, and has laid the groundwork for the evaluation of the potency of a vaccine against EL using a vaccination and challenge experiment.

A 3.5-year-old Thoroughbred mare presented at necropsy with a large mass at the root of the mesentery and multiple smaller mesenteric masses. The mucosa of the small intestine contained numerous raised nodules. Histologic examination revealed severe granulomatous mesenteric lymphadenitis and enteritis. Epithelioid macrophages and multinucleated giant cells frequently contained numerous intracytoplasmic yeast organisms, which were strongly positive on immunohistochemical staining when using a polyclonal antibody against Histoplasma spp. A diagnosis of abdominal histoplasmosis was made based on the gross, microscopic, and immunohistochemical findings.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.

The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4(+) and CD8(+) cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-gamma, TNF-alpha, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell(-/-) mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4(+) cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on gammadelta T cells or NK cells. Elimination of Thy-1.2(+) cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell(-/-) and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.

Histoplasmosis is a fungal infection caused by the Histoplasma capsulatum. Mucosal manifestations are uncommon, but, when present, they are usually associated with pulmonary or chronic disseminated infection. The course of the disease is often related to the host immune response. The purpose of this article is to describe the clinical and microscopic findings of unusual involvement of nasal cartilage and septal destruction, and oral lesions of histoplasmosis in an immunosuppressed patient who presented an unusual form of the disease.

Histofarcin, a skin test antigen for the diagnosis of epizootic lymphangitis, was locally produced from the mycelial form of Histoplasma capsulatum var. farciminosum (HCF) in disease-endemic districts of Ethiopia and tested for its application in the field between April 2002 and May 2003. The test was evaluated using 108 mules, 84 in endemic and 24 in disease-free districts. Microscopic and mycological examinations of clinical lesions were used as the "gold standard" for the validation of the test. The concentration of histofarcin that caused an optimum reaction was 0.2-0.4 mg/mL in a 0.1 mL dose and this was attained 24-48 h post-injection. The sensitivity and specificity of the histofarcin test were 90.3% (CI = 73.1, 97.5) and 69% (95%, CI = 48.1, 84.9%) in disease-endemic districts. On the other hand, specificity was 100% (CI=94.8, 100) in disease-free districts. Positive and negative predictive values of the histofarcin test were 77.78% (95% CI = 60.4, 89.3) and 85.71% (95% CI = 62.6, 96.2), respectively. A marginal substantial agreement (kappa = 0.61, P = 0.0000) was observed between the clinical status and the result of the histofarcin test. A large proportion (31%) of 'false positives' was recorded in endemic districts, which could be due to the pre-clinical stage of the disease. The latter ended in lower specificity and positive predictive value of the test since the true infection status of a 'false positive' could not be known on the basis of clinical features. Therefore, standard test validation procedures including slaughtering and isolation of HCF is required. After proper validation, we conclude that the histofarcin test could play a significant role in detecting early infection, and differentiating of EL from glanders, ulcerative lymphangitis, and sporotrichosis.

Histoplasma capsulatum is a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytose H. capsulatum yeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC kill Histoplasma is via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mphi, Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin to Histoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation of Histoplasma-infected DC at 18 degrees C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degrade H. capsulatum yeasts. Further, culture of Histoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasma activity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture of Histoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasma activity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used by H. capsulatum yeasts to survive intracellularly within Mø.

Histoplasma capsulatum (Hc) is a dimorphic fungal pathogen indigenous to the Ohio and Mississippi River Valleys in the United States. Infection is initiated by inhalation of microconidia or small mycelial fragments into the terminal bronchioles of the lung. The conidia are taken up by alveolar macrophages (Mphi), in which they convert to the pathogenic yeast phase. The yeasts replicate in the alveolar Mphi and other Mphi recruited to the lung as part of the inflammatory response. Thus, the yeasts are able to disseminate from the lung to other organs, such as the liver and spleen. As a facultative intracellular parasite, the interaction of Hc yeasts with Mphi is a critical component of the host response to infection. In addition, Hc yeasts have critical interactions with inflammatory neutrophils, and with dendritic cells (DCs) in the lung and other organs. Indeed, recent new evidence suggests that DCs may be the key antigen-presenting cells that initiate cell-mediated immunity. Thus, the methods described in this chapter cover quantitation of the binding, ingestion, and intracellular replication of Hc yeasts in human Mphi, DCs, and neutrophils.

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.