Yeast Phase Of Histoplasma Capsulatum Research Articles

Biological Function and Molecular Mapping of M Antigen in Yeast Phase of Histoplasma capsulatum

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

Diagnosis of histoplasmosis in immunosuppressed patients

To define the most appropriate studies for making a diagnosis of histoplasmosis in immunosuppressed patients. As is true of all fungal infections in immunosuppressed patients, heightened awareness of the epidemiology and clinical manifestations of histoplasmosis is essential in making an early diagnosis. Increasingly, Histoplasma antigen detection is used to help establish a diagnosis of histoplasmosis. Most of the reported data are on patients with AIDS, but limited data suggest the usefulness of this assay in other immunosuppressed patients as well. False positive reactions occur with other fungal infections, especially blastomycosis, and patients who have histoplasmosis may have a false positive serum Aspergillus galactomannan assay. The identification of the yeast phase of Histoplasma capsulatum in tissue biopsy samples and, uncommonly, in circulating blood phagocytes is also helpful in establishing a diagnosis quickly. PCR techniques have yet to prove useful for the rapid diagnosis of histoplasmosis, and serology is often negative in immunosuppressed patients. Culture remains definitive and should always be performed to confirm the results of the rapid diagnostic studies. Rapid techniques, mostly antigen detection in serum and urine and histopathological identification of Histoplasma capsulatum in tissues, are the most important rapid diagnostic tests for histoplasmosis in immunosuppressed patients.

Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism.

A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil. We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium-binding protein (CBP). Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium-binding sites. We also evaluated CBP1 expression by reverse transcription-polymerase chain reaction (RT-PCR) of yeasts grown in broth culture and within two host cell types, a macrophage-like cell line and respiratory epithelial cells. H. capsulatum yeasts expressed CBP1 in all of these settings. Splenocytes from mice immunized with H. capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts. In addition, after H. capsulatum yeasts were subjected to a calcium-free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP. These results suggest that CBP may function to provide yeasts with calcium when they are in a low-calcium environment, such as the phagolysosomal compartment within macrophages.

Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice

The incorporation of (3H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum. The ability to incorporate (3H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased. Incorporation of (3H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted. Results showed that inoculation of H. capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.

Treatment of disseminated histoplasmosis in hamsters

A comparative study between itraconazole, ketoconazole and amphotericin B in the treatment of experimental histoplasmosis in hamsters was carried out. Seventy five animals were inoculated intracardiacally with the yeast-phase of Histoplasma capsulatum. They were divided in 5 groups: 1) treated with itraconazole by gavage (g) at a daily dose of 16 mg/kg; 2) treated with ketoconazole by (g) at a daily dose of 80 mg/kg; 3) treated with amphotericin B intraperitoneally (i.p.) at 6 mg/kg every other day; 4) control animals receiving distilled water i.p. and 5) control animals receiving P.E.G. 200 by (g). All the treatments were started one week after the challenge inoculation and they were given for 21 days. The results were evaluated by autopsy of all the animals one week after the end of the treatments. The following determinations were taken into account: microscopic examinations of spleen, liver and lungs and cultures of the spleen with determination of colony forming units/g. All the antifungal drugs used in this study were able to cause negative microscopic examinations of the liver, spleen and lungs; but only amphotericin B produced culture negative results. Itraconazole and ketoconazole presented 66% and 86% of positive cultures respectively, nevertheless the C.F.U. were lower than those obtained in control groups. In these experimental conditions amphotericin B seems to be more active than the azolic compounds and itraconazole is slightly superior to ketoconazole at a lower dose.

Quantitative plating ofHistoplasma capsulatumwithout addition of conditioned medium or siderophores

We have formulated two defined media which yield excellent plating efficiency of the yeast phase of Histoplasma capsulatum. Neither requires the addition of conditioned medium or purified siderophores, although the simpler of the two media includes hemin as a source of solubilized iron. Agarose, rather than conventional agar, is the solidifying agent for both media. Plating efficiency usually exceeds 90% with both North American and Central American isolates of H. capsulatum.

Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum.

Five alkali-stable lipids from the yeast phase of Histoplasma capsulatum have been purified and analyzed. Each compound has equimolar amounts of hydroxysphinganine (phytosphingosine) and a hydroxy or nonhydroxy 24:0 fatty acid. All yield inositol phosphate after acid hydrolysis, and several are novel in that they also yield dimannosylinositol (compound V) and isomeric galactosyldimannosylinositols (compounds VI and VIII) after strong ammonolysis. The foregoing as well as other data suggest that compound V is a dimannosylinositolphosphoceramide and compounds VI and VIII are galactosyldimannosylinositolphosphoceramides with isomeric head groups. The chromatographic behavior of compounds II and III indicates that they are similar to the inositolphosphoceramides previously observed in Saccharomyces cerevisiae. Compounds V and VI are virtually absent from the mycelial phase of H. capsulatum. Antibodies that react with compounds V, VI, and VIII have been detected in sera from patients with histoplasmosis.

Purification and characterization of a cysteine dioxygenase from the yeast phase of Histoplasma capsulatum.

A cysteine dioxygenase, cysteine oxidase (EC 1.13.11.20), has been purified from the cytosolic fraction of yeast phase cells of the dimorphic fungus Histoplasma capsulatum. The cysteine oxidase is an iron-containing dioxygenase with a molecular weight of 10500 (+/- 1500) and is present only in the yeast phase of the fungus. The enzyme is highly specific for L-cysteine, with a Km of 2 X 10(-5) M in vitro. The product of cysteine oxidation is cysteinesulfinic acid, as analyzed by thin-layer chromatography and mass spectroscopy. To our knowledge, this is the first cysteine oxidase isolated from a fungus, and it probably plays an important role in the mycelial to yeast phase transition of H. capsulatum during which redox potential and cysteine levels are crucial factors.

Estudio de dos antigenos de la fase levaduriforme delHistoplasma capsulatumpara pruebas cutaneas

The results of skin tests with two antigens of the yeast phase of Histoplasma capsulatum are presented. Both antigens were able to fix complement and form precipitating bands in the presence of sera from patients with active histoplasmosis. Their sensitivity was lower than the metabolic antigen usually employed in serology. Results of skin tests obtained with both yeast phase antigens and a standard histoplasmin coincided in animals infected with H. capsulatum. Only one cross reaction was observed in animals inoculated with Paracoccidioides brasiliensis. Skin tests in humans were conducted on patients with and without mycotic disease. The percentages of positive reactions in patients with histoplasmosis were not significantly different between control histoplasmin and whole cell extracts. In the patients with non mycotic diseases the frequency of positive tests varied between 28.8% and 32%, which agrees with previous statistical data for the general population of the area. Equally the positive percentage of 55% and 68% in patients with paracoccidioidomycosis coincided with the results of previous studies. The histological patterns of these skin tests showed that they were produced by cell mediated hypersensitivity. The sensitivity of a whole yeast cell extract was similar to histoplasmin L48, but its preparation was quicker and easier to perform and it had no foreign substances from the culture medium, so we think that it would be easier to standardize it chemically.

Inhibition of Histoplasma capsulatum ribonucleic acid polymerases by homologous and heterologous ribonucleic acid

The ribonucleic acid (RNA) polymerases from the yeast phase of Histoplasma capsulatum are differentially sensitive to RNA isolated from the yeast and mycelial phases of this fungus and from Escherichia coli. Low-molecular-weight RNA from H. capsulatum was the most effective inhibitor.

Ribonucleic acid polymerases of the yeast phase of Histoplasma capsulatum.

Ribonucleic acid (RNA) polymerases of Histoplasma capsulatum (yeast phase) were fractionated by phosphocellulose chromatography and partially characterized. Three distinct, active fractions were seen. The major RNA polymerase species was inhibited strongly by alpha-amanitin, whereas the other two were resistant. When either slightly purified (HSE) extract or the major active component was assayed at 37 C, the incorporation of tritiated uridine monophosphate into RNA stopped after 10 to 15 min. In contrast, the synthesis continued for at least 1 h at 23 C. The other two RNA polymerase species exhibited higher rates of incorporation when tested at 37 C, and continued to synthesize RNA even after 60 min. However, by that time the levels of incorporation at 23 C were higher than at 37 C for all three enzymes. The temperature sensitivity was not affected by changing substrate concentration or employing either native or denatured calf thymus deoxyribonucleic acid as a template. These results are compared with the data obtained with RNA polymerases from different fungi and other organisms. A possible involvement of RNA polymerase(s) in morphological differentiation of H. capsulatum is discussed.

Isolation of a new soluble antigen from the yeast phase of Histoplasma capsulatum.

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.

Evaluation of various media for growth of selected pathogenic fungi and Nocardia asteroides.

Growth of five strains of mycelial and yeast phases of Sporothrix schenckii and Blastomyces dermatitidis , five strains of mycelial and three strains of yeast phases of Histoplasma capsulatum , five strains of mycelial phase of Coccidioides immitis , and five strains each of Nocardia asteroides and Cryptococcus neoformans was evaluated on brain heart infusion (BHI), heart infusion (HIA), and trypticase soy (TSA) agars containing human and sheep blood at concentrations of 5 and 10%. BHI and HIA were equally efficient in supporting growth of the organisms tested. Varying the amount of blood did not have an appreciable effect on growth. For the conversion or maintenance of the yeast phases of the dimorphic fungi, human blood was more efficient than sheep blood; however, in all other aspects studied they appeared to be comparable. TSA with sheep or human blood was clearly inferior to the other media studied.

Amphotericin B potentiation of rifampicin as an antifungal agent against the yeast phase of Histoplasma capsulatum.

Rifampicin, at high concentrations, inhibited growth and RNA synthesis in the yeast phase of Histoplasma capsulatum. These effects were potentiated by low concentrations of amphotericin B. The combination of the two agents was fungicidal, whereas each alone, at much higher concentrations, was only fungistatic.

Regeneration of Protoplasts of Histoplasma Capsulatum: A Study by Light Microscopy

Protoplasts of the yeast phase of Histoplasma capsulatum prepared by incubation in Sabouraud broth + 1.5 M MgSO4 +2-deoxy-d-glucose will regenerate to the sporulating mycelial phase in a variety of osmotically supportive media at 25 C, and to the budding yeast phase at 37 C. Freshly formed and isolated protoplasts are osmotically fragile. They become resistant to osmotic shock within a few hours at 4 C and remain viable and capable of regeneration after more than 2 months at -70 C. Methods of protoplast induction, separation and concentration under sterile conditions are described.

Factors affecting mycelial to yeast phase conversion and growth of the yeast phase of Histoplasma capsulatum.

A procedure for rapid induction of mycelial to yeast phase (M→Y) conversion ofHistoplasma capsulatum has been devised. Exposure of mycelial fragments to low oxidation-reduction (O/R) potentials (+5 to+65 mv) either aerobically (ascorbic acid treatment) or anaerobically (nitrogen atmosphere) for 18 to 24 hours at 37° C resulted in induction of the M→Y conversion process whether or not an organic sulfur source was available. However, aerobic conditions and a suitable organic sulfur source, such as cysteine, cystine or lanthionine were found essential for outgrowth and maintenance in the yeastlike phase.

Isolation of a New Soluble Antigen from the Yeast Phase of Histoplasma capsulatum

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.

Studies on Protoplast Induction in the Yeast Phase of Histoplasma Capsulatum by Magnesium Sulfate and 2-Deoxy-D-Glucose

Studies on Protoplast Induction in the Yeast Phase of Histoplasma Capsulatum by Magnesium Sulfate and 2-Deoxy-D-Glucose

Studies on Protoplast Induction in the Yeast Phase of Histoplasma capsulatum by Magnesium Sulfate and 2-Deoxy-D-Glucose

SUMMARYExperiments to elucidate the possible mode of action of MgSO4 with or without 2-deoxy-d-glucose (2DG) in inducing protoplasts in early log phase yeast cells of Histoplasma capsulatum Darling without added external enzymes are described and discussed. MgSO4 is effective in a molarity range between 1.8 and 2.2 M. Below 1.8 M no protoplasts are formed, while 2.4 M MgSO4 produces irreversible plasmolysis. For acceleration of protoplast formation, the optimum concentration of 2DG is 750 mg/100 ml medium. Protoplasts are formed at glucose concentrations ranging from 0.1% w/v to 1.25% w/v. Glucose concentrations of 0.1% w/v occasionally yield multiple budding whole yeast cells.

Characterization of antigens from the yeast phase of Histoplasma capsulatum.

Preliminary studies established methods for obtaining maximum yields of viable cells. Liquid shaken cultures of Histoplasma capsulatum provided a maximum of 4 x 10(8) to 5 x 10(8) cells/ml regardless of the inoculum size. Under optimum conditions, cells were viable (90 to 95%) for 168 to 240 hr. Generation times ranged from 7.52 to 8.36 hr. Immunodiffusion, immunoelectrophoresis, and ultracentrifugation studies on phenol and ethylenediamine extracts of intact cells and cell walls revealed the presence of two components in the ethylenediamine extracts and three in the phenol preparations. The ethylenediamine extracts from intact cells and cell walls seemed to be identical although one of the components was more abundant in cell walls. Mice injected intraperitoneally with intact cells or cell walls were protected against intravenous challenge with H. capsulatum. Among the extracts, the water-soluble ethylenediamine extract from cell walls was most immunogenic. The other extracts gave only a light protection or none at all. Intact cells and cell walls were slightly toxic to mice. Two of the extracts were toxic when incorporated into Freund's complete adjuvant.