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PubMed2006

Characterization of the Histoplasma capsulatum-induced granuloma.

Rising rates of Histoplasma capsulatum infection are an emerging problem among the rapidly growing population of immune-compromised individuals. Although there is a growing understanding of systemic immunity against Histoplasma, little is known about the local granulomatous response, which is an important component in the control of infection. The focus of this article is the characterization of Histoplasma-induced granulomas. Five days after i.p. infection, infected macrophage appear in the liver and lung; however, no granulomas are apparent. Two days later, well-formed sarcoid granulomas are abundant in the lung and liver of infected mice, which contain all visible Histoplasma. Granulomas are dominated by macrophage and lymphocytes. Most of the Histoplasma and most of the apoptotic cells are found in the center of the lesions. We isolated liver granulomas at multiple time points after infection and analyzed the cellular composition, TCR gene usage, and cytokine production of granuloma-infiltrating cells. The lesions contain both CD4+ and CD8+ T cell subsets, and T cells are the primary source of IFN-gamma and IL-17. The main source of local TNF-alpha is macrophage. Chemokines are produced by both infiltrating macrophage and lymphocytes. Dendritic cells are present in granulomas; however, T cell expansion seems to occur systemically because TCR usage is very heterogeneous even at the level of individual lesions. This study is the first direct examination of host cellular responses in the Histoplasma-induced granuloma representing the specific interface between host and pathogen. Our studies will allow further analysis of key elements of host Histoplasma interactions at the site of chronic infection.

Animals↗
PubMed1992

In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds.

Base Sequence↗
PubMed1968

Inhibition of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar media.

The inhibition of growth of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar was investigated. Histoplasma (yeast-phase inoculum) was grown alone and in mixtures with yeasts at 25 C for 4-week periods. As few as 10 colonies of C. albicans completely inhibited the growth of approximately 50,000 potential colonies of Histoplasma. The pH was determined in cultures of 36 colonies of Candida on media containing 1, 2, and 4% glucose by spotting the agar with pH indicators. A drop in the pH became noticeable in all three media about the 3rd day of incubation, and a pH of 3.5 was reached in about 7 days. Subsequently, the pH remained almost stationary in the 4% glucose-agar, rose slowly in the 2% glucose-agar, and rose sharply in the 1% glucose-agar. The growth of Histoplasma was inhibited completely at pH 4 and below. When the pH was controlled in mixed cultures, some growth of Histoplasma was obtained. Substitution of maltose for glucose delayed the development of acidity and allowed the appearance of numerous mycelial colonies in the presence of Candida. This growth was arrested as soon as the medium became acid. Four other species which also acidified the Sabouraud's medium effected similar inhibition. It was thus shown that severe and prolonged acidity produced by some yeasts in the sugar-rich Sabouraud's media is alone sufficient to completely inhibit Histoplasma during the standard 4-week incubation of specimens such as sputum.

Candida↗
PubMed1990

Histoplasma capsulatum endophthalmitis after cataract extraction.

A 60-year-old white man from an area endemic for Histoplasma capsulatum presented with a vitreous wick, hypopyon iritis, and dense vitreitis 2 months after removal of an anterior chamber intraocular lens (IOL) for chronic iritis. A diagnostic vitrectomy was performed and H. capsulatum was cultured and identified 2 weeks later. Despite intravitreal and intravenous amphotericin as well as repeat vitrectomies, the inflammation worsened and the eye was removed. Results of histopathologic examination showed Histoplasma organisms along the vitreous wick, over the surface of the iris and ciliary body, and over the retina. No organisms were found in the choroid. Dalen-Fuchs-type nodules similar to those of sarcoid also were noted, but there was no evidence of granulomatous inflammation in the uvea. Because of his unilateral disease with Histoplasma in the vitreous wick, negative serology, and an absence of systemic infection, the authors believe that this patient had a previously unreported form of ocular Histoplasma, exogenous postoperative Histoplasma endophthalmitis.

Cataract Extraction↗
PubMed2000

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.

Animals↗
PubMed1993

Histoplasma capsulatum modulates the acidification of phagolysosomes.

The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.

Animals↗
PubMed2004

RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence.

Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.

Genes, Reporter↗
PubMed1997

In vitro interactions between Histoplasma capsulatum var. duboisii and other fungi.

Histoplasma capsulatum var. duboisii has only recently been shown to occur naturally in soil admixed with bat guano in a bat cave. We report our observations on the in vitro interaction of Histoplasma capsulatum var. duboisii with other fungi recovered from the same habitat or other sources by employing co-culture technique and by investigating antibiosis between H. capsulatum var. duboisii and other fungi. The range of interactions varied from total overgrowth of Histoplasma by Aspergillus fumigatus and Microsporum gypseum, through partial antagonism by Lecythophora mutabilis, to mutual inhibition by Wangiella dermatitidis. Those fungi that exhibited a competitive advantage in terms of growth did not affect the viability of Histoplasma. Lysis and mycoparasitic structures were not observed in the co-cultures. However, the cultural filtrate of Chrysosporium indicum inhibited the growth of the Histoplasma strains. The results suggest the ability of H. capsulatum var. duboisii to co-exist with a variety of other potentially pathogenic and non-pathogenic fungi in an ecological niche.

Animals↗
PubMed1989

Significance of Histoplasma antigen in the cerebrospinal fluid of patients with meningitis.

A radioimmunoassay was previously developed for detection of Histoplasma capsulatum antigen in the blood and urine of patients with disseminated histoplasmosis. In this investigation, cerebrospinal fluid (CSF) specimens from 14 episodes of Histoplasma meningitis occurring in 12 patients were tested by radioimmunoassay. Histoplasma capsulatum antigen was detected in the CSF of five patients. Cerebrospinal fluid cultures were positive for H capsulatum in three of these five patients. Antibodies to H capsulatum were found in nine of the 13 CSF specimens tested. The radioimmunoassay for Histoplasma antigen was also positive in the CSF in one of 11 patients with coccidioidal meningitis but not in 17 patients with cryptococcal meningitis. It was concluded that Histoplasma antigen is present in the CSF of some patients with histoplasmosis and chronic meningitis, but cross-reactions may occur in patients with coccidioidal meningitis.

Antigens, Fungal↗
PubMed1977

Histoplasma meningitis with common variable hypogammaglobulinemia.

Histoplasma meningitis (HM) has been reported to occur primarily in association with disseminated histoplasmosis (DH). We report a case of histoplasma meningitis occurring in a patient with common variable hypogammaglobulinemia (CVH) in which no manifestations of DH were observed. L. L., a 66-year-old Caucasian male, clerical worker, developed occasional episodes of dizziness and tinnitus in mid-1971. During 1972, increasing frequency of these episodes and gradually progressive confusion were noted. In January 1973, vomiting, forther confusion, obnubilation, and a left central facial paresis developed and he was hospitalized. Physical examination revealed no pulmonary abnormalities, lymphadenopathy or hepatosplenomegaly. Over the ensuing 6-week evaluation, there was occasional fever to 38.5 degrees C. Chest roentgenogram was normal. Cerebral angiography suggested a mass in the left cerebellar hemisphere. EEG was diffusely slow. Multiple CSF examinations revealed: Glucose 7-18 mg/with a normal blood glucose, protein 109-256 mg/and cells 66-140 (95 + % mononuclear). Histoplasma capsulatum was cultured from CSF but not from sputum, urine, blood or bone marrow. Skin tests for PPD, histoplasmosis, coccidiodomycosis, blastomycosis, mumps, dinitrochlorobenzene and streptokinase-streptodornase were negative then and 6 months later. Histoplasma serum antibody was absent. Immunoglobulin analysis revealed IgG 430 mg %, IgA 46 mg %, and IgM 35 mg %, which with the history and skin test results suggested CVH. Treatment with 2.51 gm of amphotericin B given intravenously over a 3-month period resulted in complete reversal of all neurologic signs and clearing of the confusion. The remission has been maintained for two years. This case represents a primary infection of the CNS by histoplasma. The relationship between the HM and the CVH will be discussed.

Agammaglobulinemia↗
PubMed1976

Specific immunological test for the rapid identification of members of the genus Histoplasma.

A sensitive and specific immunological method was developed for rapid identification of the mycelial forms of Histoplasma capsulatum var. capsulatum, H. capsulatum var. duboisii, and H. farciminosum and for separation of these pathogenic fungi from morphologically similar hyphomycetes and other fungal pathogens. This method is based on the fact that all of the Histoplasma spp. produce H and M histoplasmin antigens, whereas the other fungi do not. Inocula consisting of heavy mycelial growth from a pure, full-grown culture were transferred into flasks containing small volumes of brain heart infusion broth. These cultures were placed on a shaker and grown at 25 C. Using the micro-immunodiffusion technique and antisera containing antibodies to H and M precipitinogens, we detected exoantigens in 3-day-old brain heart infusion culture supernatants concentrated 25 and 50 times. The ability of the procedure to identify Histoplasma spp. was evaluated by testing 96 unknown mycelial cultures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown were tested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratinophilum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedonium sera and human histoplasmosis case serum. Each unknown was also identified by conventional laboratory procedures involving cultural and, where necessary, in vivo studies. In the comparative evaluation the immunological test was observed to be 100% sensitive. It permitted the accurate generic identification of the Histoplasma spp. within 5 days, in contrast to the average of 33 days required by the routine mycological procedure.

Antigens, Fungal↗
PubMed1978

Histoplasma meningitis with hyperactive suppressor T cells in cerebrospinal fluid.

Histoplasma meningitis usually occurs in conjunction with disseminated histoplasmosis. We studied a patient with common variable hypogammaglobulinemia who manifested meningitis without disseminated histoplasmosis. No histoplasma antibody was detected in cerebrospinal fluid (CSF) or blood. Evaluation of lymphocyte function in the blood revealed normal numbers of T cells with increased numbers of B cells. Most blood lymphocytes were identifiable, but most lymphocytes in CSF were null cells. Lymphocyte proliferative response to phytohemagglutinin or pokeweed mitogen was poor. T cells in CSF suppressed proliferative responses to histoplasma antigen by cells from blood or CSF, whereas T cells from blood did not. This difference suggested compartmentalization of T-cell function. The lack of humoral and cellular response to histoplasma in CSF may have allowed meningitis to develop, while the cellular response to histoplasma elsewhere prevented development of disseminated histoplasmosis.

Aged↗
PubMed2003

Phylogeography of the fungal pathogen Histoplasma capsulatum.

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.

Cluster Analysis↗
PubMed2002

Molecular cell biology and molecular genetics of Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic ascomycete which is capable of producing a broad spectrum of disease ranging from mild asymptomatic, pulmonary illness to severe, life-threatening systemic mycosis. Regulatory mechanisms that use temperature and other environmental cues are paramount to the successful adaptation of the organism as an effective intracellular pathogenic yeast. Although the biochemistry and phenomenology of reversible morphogenesis have been well examined in Histoplasma, the identification and functional characterization of genes and their products that are required for early establishment or maintenance of the parasitic yeast phase in intracellular host compartments have only recently been fruitful. Advances in the molecular biology of Histoplasma, including approaches to introduce telomeric plasmids, reporter fusion constructs, and gene disruption cassettes into the fungus are poised to solidify the pre-eminence of this fungus as a model system which can be applied to other dimorphic fungal pathogens that exhibit similar cellular and immunological complexities. This review centers on recent developments in the molecular cell biology and molecular genetics of Histoplasma capsulatum that provide important new avenues for examining the mold-to-yeast phase transition beyond the historical, binary view of dimorphism and the implications that these successful approaches may have on seminal issues in fungal pathogenesis.

Gene Expression Regulation, Fungal↗
PubMedDate not supplied

Cloning and analysis of an actin-encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum.

We have cloned an actin-encoding cDNA from the dimorphic fungus Histoplasma capsulatum, an important pathogen of humans. The predicted amino acid sequence as well as the general codon pattern of Histoplasma actin revealed the highest degree of similarity to the actin of the filamentous ascomycete Aspergillus nidulans. Southern blot analysis determined that actin was encoded by a single copy in the Histoplasma genome. Northern blot analysis showed a single 1700 nt transcript in yeast and mould cells as well as in cells undergoing the temperature induced mould-to-yeast conversion. Actin mRNA levels normalized to 18 S rRNA were found to be equivalent in all the stages examined, except for a sharp four-fold transient decrease 4 h into the mould-to-yeast conversion. These data suggest that actin mRNA would not be a suitable internal marker for expression studies during Histoplasma mould-to-yeast morphogenesis.

Actins↗
PubMed2005

The combined effect of IL-4 and IL-10 suppresses the generation of, but does not change the polarity of, type-1 T cells in Histoplasma infection.

Dominant type-1 cytokine production is induced in a murine model of systemic histoplasmosis. We used this model to investigate whether the presence of antagonistic cytokines during T cell priming changes the polarity of T cells in response to Histoplasma infection. Before infection with Histoplasma capsulatum, mice were injected twice with goat anti-mouse IgD antiserum (GalphaMdelta), which induced expression of dominant type-2 cytokines. At days 7 and 14 after infection, the GalphaMdelta-treated mice had suppressed IFN-gamma response and a significantly greater fungal burden in their spleens and lungs. The number of IFN-gamma-producing cells as well as the level of IFN-gamma produced per cell was greatly reduced. Not only CD4+ T cells but also CD8+ T cells were affected. The number of Histoplasma-induced IFN-gamma-producing cells was partially restored in GalphaMdelta-treated IL-4-/- and IL-10-/- mice and completely restored in IL-4-/- IL-10-/- mice. Thus, the combined effect of IL-4 and IL-10 suppressed the generation of IFN-gamma-producing cells. A longitudinal study demonstrated that as IL-4 and IL-10 decreased, the number of Histoplasma-induced IFN-gamma-producing cells rapidly increased, and fungal clearance improved, demonstrating that the presence of IL-4 and IL-10 did not permanently change the polarity of T cells.

Animals↗
PubMed1996

Hepatic granulomas in children. A clinicopathologic analysis of 23 cases including polymerase chain reaction for histoplasma.

In a 15-year period at the Riley Hospital for Children, granulomas were found in 23 (4%) of a total of 521 liver biopsies. An etiology was identified in 87%: Histoplasma was diagnosed in 15 cases (65%) by polymerase chain reaction (PCR) on paraffin-embedded tissue, serology, and special stains; sarcoidosis was diagnosed in four cases; and schistosomiasis was diagnosed in one case. Serial liver biopsies were available from five patients; granulomas occurred in only one biopsy of the series from each patient. Extrahepatic tissue from six patients contained granulomas, and an etiology for the liver granulomas was identified in all six patients (four histoplasmosis, two sarcoidosis). The extrahepatic tissue from two patients with Histoplasma was diagnostic. We made the following conclusions: that PCR is applicable to archival material and greatly increases the yield of specific infectious diagnoses of liver granulomas compared with conventional diagnostic methods (65 versus 22%); that the infections causing liver granulomas are those that are endemic in a community (e.g., Histoplasma in Indiana); that Histoplasma can coexist with a wide variety of systemic and primary liver diseases; that the likelihood of identifying a cause of liver granulomas is increased if there are extrahepatic granulomas; and that hepatic granulomas may have a limited life span. Treatment of liver granulomas should be determined by the clinical setting and directed at the underlying cause.

Adolescent↗
PubMed1998

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.

Drug Resistance, Microbial↗