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PubMed1994

Clearance of Histoplasma capsulatum variety capsulatum antigen is useful for monitoring treatment of experimental histoplasmosis.

We sought to determine whether measurement of Histoplasma capsulatum var. capsulatum antigen concentration in tissues and blood provided a marker for antifungal effect of itraconazole in a nonlethal murine model of histoplasmosis. Treatment with itraconazole (Sporanox), in cyclodextrin, was evaluated in a pulmonary model of histoplasmosis. Mice infected with 4.0 x 10(7) yeast-phase organisms by endotracheal inoculation were treated with itraconazole, 1.5 mg twice daily by gavage, for 10 consecutive days, beginning on day 4 of infection. All mice were sacrificed on day 15 of infection. Blood, spleen, and lung tissues were removed for culture and quantification of antigen. Numbers of organisms were significantly lower in spleens from the treated group: 20.8 +/- 41.8 vs. 65.8 +/- 39.1 in the control group, P = 0.017. Numbers of organisms in lung were 9.6 +/- 27.3 colony forming units in treated versus 24.2 +/- 36.3 in control animals, P = 0.267. Antigen concentrations in spleen tissue and serum were lower in treated versus control mice: spleen, 1.8 +/- .6 units in treated versus 11.0 +/- 2.3 in controls, P < 0.001; serum, 0.8 +/- 0.2 units in treated versus 2.2 +/- 1.0 units in controls, P < 0.001. Lung antigen concentrations were similar in the two groups, 19.2 +/- 1.4 units in treated compared to 17.9 +/- 3.0 units in control mice, P = 0.142. The cyclodextrin formulation of itraconazole (Sporanox) demonstrated antifungal activity in experimental histoplasmosis. Antigen detection was a useful marker for antifungal effect.

Animals↗
PubMed1988

Phagosome-lysosome fusion in P388D1 macrophages infected with Histoplasma capsulatum.

The issue of whether or not phagocytized Histoplasma capsulatum yeasts evade phagosome-lysosome fusion (P-LF) has been debated by several investigators. To resolve this problem, yet avoid drawbacks associated with the conventional assays of P-LF (electron microscopy and the acridine orange assay), we used fluorescein isothiocyanate-labeled dextran (FITC-dextran) to monitor P-LF in the macrophage-like cell line P388D1.D2. Controls indicated that FITC-dextran could be used to distinguish between evasion of P-LF by Toxoplasma gondii and phagolysosome formation following ingestion of Saccharomyces cerevisiae. Phagosomes containing H. capsulatum clearly fused with FITC-dextran-labeled lysosomes at a rate comparable to that observed for S. cerevisiae. This was true for several strains of H. capsulatum including two avirulent strains derived in this laboratory. Varying the dose of H. capsulatum did not alter the percentage of phagolysosomes formed. Our results indicate that H. capsulatum is one of a small number of organisms which is able to survive in phagolysosomes.

Acridine Orange↗
PubMed1989

Requirements for histoplasmin presentation by accessory cells to a Histoplasma capsulatum-reactive T-cell line.

We examined the pathways involved in presentation of native histoplasmin by adherent splenocytes (as a source of accessory cells) to JC1, a Histoplasma capsulatum-reactive murine T-cell line that is CD4+. JC1 did not respond to accessory cells that had been fixed with paraformaldehyde and then exposed to histoplasmin but did proliferate to antigen-pulsed cells that were subsequently fixed. Accessory cells that were coincubated with histoplasmin and sodium azide or 2-deoxy-D-glucose failed to induce proliferation of JC1. Moreover, accessory cells exposed to the lysosomotropic agents, chloroquine and ammonium chloride, were unable to present antigen. Monensin also inhibited presentation of histoplasmin if added to accessory cells concomitant with antigen. In contrast, accessory cells that had been pulsed with antigen for 2 hr and then exposed to each inhibitor for 2 hr stimulated proliferation of JC1. The antigen-presenting capacity of accessory cells that had been pulsed with histoplasmin for 2 hr was diminished considerably by subsequent treatment with phospholipase A2. Additional studies demonstrated that cerulenin, which depresses posttranslational lipid modification of proteins, abolished presentation of histoplasmin. The reactivity of JC1 was sharply reduced by anti-L3T4 (CD4) or by anti-I-Ab monoclonal antibody. The results not only indicate that presentation of histoplasmin requires active metabolic events within accessory cells, they also delineate the pathways involved in handling this antigen.

Ammonium Chloride↗
PubMed1992

Recognition of Histoplasma capsulatum yeast-cell antigens by human lymphocytes and human T-cell clones.

We have prepared a detergent extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells that is recognized by T cells from mice immunized with viable organisms or with the extract. A 62-kd antigen from this extract has been isolated and has been shown to be antigenic and to confer protective immunity in mice. In this study, we examined the in vitro proliferative response by human lymphocytes and human T-cell clones to both the extract from the cell wall and cell membrane and the 62-kd antigen, termed HIS-62. Seven healthy individuals were identified whose peripheral blood mononuclear cells responded to the cell wall and cell membrane extract from H. capsulatum yeast cells. Mononuclear cells from all seven individuals recognized histoplasmin. T-cell clones were generated from peripheral blood mononuclear cells from a single person using the extract from cell wall and cell membrane as the antigenic preparation. Nineteen clones were propagated; all expressed the surface phenotype CD3+, CD4+, T-cell receptor alpha/beta +. The clones were antigen-specific. Of 17 clones studied, none responded to extracts from Blastomyces dermatitidis or Coccidioides immitis or to tetanus toxoid. Sixteen of 19 clones recognized HIS-62 in vitro. Additional analysis revealed that cells from each of the seven individuals who responded to the extract mounted a proliferative response to HIS-62. Thus, the extract from the cell wall and cell membrane and HIS-62 are targets of the human cell-mediated immune response to H. capsulatum. Moreover, HIS-62 appears to be an immunodominant antigen.

Adult↗
PubMed2003

Characterization of an alternative oxidase activity of Histoplasma capsulatum.

Histoplasma capsulatum possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consuming activities. The latter, carried out by a single subunit enzyme termed 'alternative oxidase', is the focus of this report. AOX1 cDNA clones were isolated and direct evidence that the cDNA ORF encodes functional alternative oxidase is reported. Also reported are the generation of an antiserum to the AOX1 protein product, and specific detection in vivo of the mRNA and protein products of the AOX1 gene. Finally, initial studies of regulation of H. capsulatum AOX1 gene expression demonstrated that RNA abundance was increased after hydrogen peroxide-mediated oxidative stress and after inhibition of mitochondrial electron transport enzymes with antimycin A or sodium cyanide. This pattern of regulation is consistent with the hypothesis that alternative oxidase contributes to survival of H. capsulatum after oxidative or metabolic stress and may be important for virulence of this pathogenic organism. The GenBank Accession Nos for the cDNA sequences reported in this paper are AF133236, AF133237 (AOX1).

Amino Acid Sequence↗
PubMed1997

An AP1 element is involved in transcriptional regulation of delta9-desaturase gene of Histoplasma capsulatum.

We have characterized a region of the promoter of a cloned delta9-desaturase gene (Ole1) of Histoplasma capsulatum, a dimorphic pathogenic fungus of humans. The product of the delta9-desaturase gene is involved in regulating membrane fluid state in animal cells and microorganisms. To identify sequences critical for Ole1 expression in both the saprobic mycelial and parasitic yeast phases of this organism, we performed a deletion analysis. Evidence is presented that a 240 nt region of the proximal promoter is involved in a phase-specific binding in vitro. By sequence analysis we have identified one likely regulatory element that coincides with an AP1 binding site (TGACTAA) that is located at -740 nt of 5'-upstream from the ATG. Using gel mobility shift assays, we show that this cis-acting element binds nuclear proteins extracted from the yeast and mycelial phases of H. capsulatum that may participate in control of expression of the delta9-desaturase gene.

Base Sequence↗
PubMed1999

An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

Amino Acid Sequence↗
PubMed2000

Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico.

The present paper represents data on the genetic polymorphism of 13 Histoplasma capsulatum isolates recovered from infected bats randomly captured in the Mexican states of Morelos, Puebla, and Oaxaca. The polymorphic DNA patterns were analyzed by two-primer RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) method. To amplify the fungal genome by PCR, the following primer arrangements were used: 5'-AACGCGCAAC-3' and 5'-AAGAGCCCGT-3'; 5'-AACGCGCAAC-3' and 5'-GTTTCCGCCC-3'; or 5'-AACGCGCAAC-3' and 5'-GCGATCCCCA-3'. A common polymorphic DNA pattern of H. capsulatum was revealed in different assays. This pattern is shared by 7 H. capsulatum isolates recovered from different specimens of nonmigratory bats (Artibeus hirsutus) captured in a cave in Morelos, by 5 isolates recovered from infected migratory bats (Leptonycteris nivalis) captured in Morelos and Puebla, and by 1 isolate from another migratory bat (L. curasoae) captured in Oaxaca. This polymorphic DNA pattern of H. capsulatum could represent fungal markers for the geographic areas studied, and considering its distribution in three different states of the Mexican Republic, the role of bats as responsible for H. capsulatum spreading in nature, in relation to their movements and migrations besides their shelter habits, is suggested. Analyses of DNA patterns of H. capsulatum isolated from infected bats, from clinical cases, and from blackbird excreta, have shown a major relatedness between bats and clinical isolates, in contrast to those isolates from bird excreta.

Animals↗
PubMed2001

Amplified single-nucleotide polymorphisms and a (GA)(n) microsatellite marker reveal genetic differentiation between populations of Histoplasma capsulatum from the Americas.

Histoplasma capsulatum has a worldwide distribution but is particularly concentrated in the midwestern United States and throughout Central and South America. Genetic differences between isolates resident in separate parts of the world have been reported, but the relationship between the isolates and the level of migration between different endemic foci has not been clear. In this study we used multilocus genotypes based on amplified polymorphic loci and one microsatellite to quantify the level of genetic differentiation occurring between North and South American populations of H. capsulatum. Significant genetic differentiation occurred between isolates obtained from Indiana and Alabama, and a marked division was seen between the Indiana population and the Class 1 isolates from St. Louis. Strong genetic differentiation occurred between the two North American populations and the Colombian population. This study supports the separation of North and South American H. capsulatum into different species, which has been proposed under the phylogenetic species concept.

Genetic Markers↗
PubMed2002

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.

Animals↗
PubMedDate not supplied

CAMP is involved in transcriptional regulation of delta9-desaturase during Histoplasma capsulatum morphogenesis.

We have characterized the promoter region of the delta9-desaturase gene from two different strains of the dimorphic fungus Histoplasma capsulatum. Desaturase transcription is regulated in the two phases of growth: it is transcribed in the yeast phase at 37 degrees C, while it is inactive in the mycelial phase at 25 degrees C. Phase transition can be induced by shifting the temperature from 25 to 37 degrees C or by adding cAMP to the growth medium. We have identified a stress-responsive cis element (STRE) responsive to cyclic AMP (cAMP)-signaling pathway and demonstrated that this element acts in H. capsulatum. We have also identified an element, hereafter called DRE (Desaturase Regulatory Element), present in the promoters of the H. capsulatum and S. cerevisiae delta9-desaturase gene. We show that this element is necessary but not sufficient to regulate transcription of the H. capsulatum delta9-desaturase gene.

Base Sequence↗
PubMed1999

Direct detection of Histoplasma capsulatum in soil suspensions by two-stage PCR.

Histoplasmosis is the most common pulmonary mycosis in the United States. The responsible fungal pathogen, Histoplasma capsulatum, grows in soils contaminated with bird or bat droppings. Inhalation of dust from contaminated areas containing H. capsulatum spores is a primary route of infection. The ability to detect H. capsulatum in soil samples has been limited by the lack of fast, reliable and inexpensive methods. A polymerase chain reaction (PCR) method was developed that allows the direct detection of H. capsulatum in soil. A two-stage PCR protocol was followed employing both fungal-specific primers and nested primers specific for the internal transcribed spacer (ITS) region of the 5.8S rRNA gene of H. capsulatum. The estimated limit of detection of this method is 10 spores. In contrast to the more expensive and indirect mouse inoculum assay, which requires 6-8 weeks for sample analysis, PCR analysis of soil contaminated with H. capsulatum can be completed in less than 2 days.

Animals↗
PubMed1998

Identification and isolation by DDRT-PCR of genes differentially expressed by Histoplasma capsulatum during macrophages infection.

Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of genes in both parasite and host. In order to identify and isolate relevant genes of the fungus, Histoplasma capsulatum, in which expression is specifically induced during its interaction with murine macrophages (Mphi), we performed a comparative analysis of the pattern of gene expression of the fungus before and after exposure to, and internalization into Mphi by using differential display reverse transcriptase-PCR (DDRT-PCR). Using a limited set of primer combinations, six cDNA fragments of H. capsulatum were identified and isolated; five representing fungal genes in which expressions were enhanced during Mphi infection, whereas one mRNA fragment was down-regulated. Slot blots followed by Northern blot analyses confirmed that the transcripts detected with cDNA clones were over expressed after 1 h of Mphi infection, whereas no transcripts were detected with mRNA purified from H. capsulatum before infection. Sequence analyses and database searches revealed no significant homology to any known sequence for five of these clones. One of the clones showed homology to the rat p105 kD protein, and to the p100 kD co-activator proteins of human and Caenorhabditis elegans. To our knowledge, this is the first experimental evidence that specific genes are differentially expressed by a fungal pathogen when it is exposed to, and phagocytosed by Mphi. Furthermore, these results show that the DDRT-PCR procedure has adequate sensitivity to detect fungal genes induced during parasite-host interaction to identify potential new targets that can be used to develop new antifungal drugs.

Amino Acid Sequence↗
PubMed2000

Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum.

Understanding how pathogens survive within the host cell is of paramount importance in the development of vaccines and therapeutic agents. This task has been particularly daunting in the study of fungal pathogens due to the lack of easily manipulated genetic systems. In recent years several molecular genetic reporter systems have been developed to identify genes expressed during the infection process and potential virulence determinants. The development of one method in particular, in vivo expression technology (IVET), has led to the discovery of several genes from various bacterial pathogens necessary for survival during infection. The recent development of molecular genetic tools for Histoplasma capsulatum has enabled us to adapt the IVET technology for this pathogenic fungus utilizing the URA5 gene, which is essential for H. capsulatum survival in mice and in cultured macrophages, as a reporter of in vivo gene expression. We report the first successful application of IVET screening of a fungal pathogen for genes expressed exclusively during infection.

Animals↗
PubMed1985

Studies on histoplasmosis farciminosi (epizootic lymphangitis) in Egypt. Isolation of Histoplasma farciminosum from cases of histoplasmosis farciminosi in horses and its morphological characteristics.

Isolation of Histoplasma farciminosum from five horses, showing typical signs of histoplasmosis farciminosi (epizootic lymphangitis) was successfully attempted. The mycelial form of H. farciminosum was isolated on Sabouraud dextrose agar enriched with 2.5% glycerol, brain heart infusion (BHI) agar enriched with 10% horse blood and PPLO dextrose glycerol agar. The last medium proved to be the most effective, both for primary isolation and subculturing of the fungus. It was found that on primary isolation, the lag phase of the mycelial form of the fungus was relatively long, involving 4-8 weeks at 25 degrees C. Colonies of the mycelial form of H. farciminosum appeared on subculture as a yellowish, light brown to deep brown, convoluted, waxy, cauliflower-like growth tending to form scant aerial growth. Conversion of the mycelial form to the yeast form of H. farciminosum was successful by subculturing either on BHI agar with 5% blood or on Pine's medium and incubating at 35-37 degrees C. Complete conversion to the yeast form was achieved only after 4-5 repeated serial transfers onto fresh media every 8 days. The yeast colonies were flat, raised, slightly or deeply wrinkled, white to light gray to grayish brown, and were pasty in consistency.

Animals↗
PubMed1990

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy.

We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5'-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/micrograms DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/micrograms DNA).

DNA Replication↗
PubMed1988

Selection and characterization of ura5 mutants of Histoplasma capsulatum.

The combined use of non-aggregating Histoplasma capsulatum strains and a defined medium which allows quantitative plating of the yeast phase has allowed us to select 5-fluoroorotic acid (5-FOA)-resistant mutants of this dimorphic fungus. Approximately two-thirds of the 5-FOA-resistant strains were auxotrophic for uracil; all were deficient in orotidine-5'-monophosphate pyrophosphorylase (OMPpase) activity. One class of OMPpase mutant (alpha), which retained a low level of OMPpase activity, was auxotrophic in the yeast phase (37 degrees C) but grew slowly in the mycelial phase (25 degrees C) without exogenous uracil. This phenotype was not due to a temperature-sensitive OMPpase activity. Both wild-type and alpha mutants had a higher OMPpase activity in the mycelial phase than the yeast phase; this increased activity may be sufficient to allow mycelial growth of alpha mutants.

Histoplasma↗
PubMed1982

Antigens from Histoplasma capsulatum and Blastomyces dermatitidis. I. Immunological comparative studies from polysaccharide-protein complexes of both fungi.

Certain groups of fungi share chemical structures which makes difficult isolation and differentiation of specific antigens by the usual methods of extraction and purification. Therefore, we have oriented our studies to the immunological and biochemical characterization of differences and similarities of molecular structures from fungi, etiologic agents of systemic mycoses, hoping to establish criteria for the utilization and handling of these antigens. A deproteinized polysaccharide-protein complex (D-PPC) was isolated from Histoplasma capsulatum and Blastomyces dermatitidis. The immunological studies with humoral tests indicate a variable cross reaction between antigens of both species. In immunodiffussion systems, the reaction was specific for each species using saline solution of phosphate butter solution, while using an agarose veronal system, the cross reactions were very evident. In addition, differences in cross reactions were obtained with immunoelectrophoresis, haemagglutination and complement fixation microtest. This variation to cross reaction responses suggest that these antigens (D-PPC) share common structures but at the same time must have some different component owned by each one of the fungi which makes them more specific than crude antigens.

Antigens, Fungal↗