Antigens from Histoplasma capsulatum and Blastomyces dermatitidis

Certain groups of fungi share chemical structures which makes difficult the 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 or phosphate buffer 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 in 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.     

Electronmicroscopy of self-parasitism by Histoplasma capsulatum and Blastomyces dermatitidis

Intrahyphal as well as intrayeast hyphae were demonstrated by electron-microscopy in bothHistoplasma capsulatum andBlastomyces dermatitidis yeastlike and mycelial phase cells grown in agitated liquid media. These structures appeared to be rather common in cultures of yeastlike cells induced to convert to the mycelial phase. In many cases there was excellent preservation of ultrastructural detail of the parasitized cell which suggested that the cell may still be viable.

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Zusammenfassung InHistoplasma capsulatum so wie auch inBlastomyces dermatitidis wurden in Hyphen und Zellen der Hefe- so wie auch der Mycelphase intracellular Hyphen durch Elektronmikrokopie nachgewiesen, wenn sie in flüssigen Schüttelkulturen gezüchtet worden sind. Diese Strukturen waren in Zellen der Hefephase, die zur Überleitung in die Myzelphase angeregt worden sind, sehr häufig. In vielen Fällen war eine sehr gute Bewahrung der ultrastrukturalen Einzelheiten der parasitierten Zelle, die es nahegelegt hat, dass die Zelle noch immer lebendig ist.

     

Inhibition of Histoplasma capsulatum and Blastomyces dermatitidis by Pseudomonas aeruginosa in vitro

Summary It was accidentally discovered thatPseudomonas aeruginosa inhibited the growth ofHistoplasma capsulatum when both of the organisms were isolated from a series of sputum specimens obtained from a single patient. Experiments were performed to determine if there is any inhibitory effect on other systemic fungi.Three fractions were obtained from a broth culture ofP. aeruginosa and their antibiotic effects tested against various systemic fungi and bacteria.Reviewed in the Veterans Administration and published with the approval of the Chief Medical Director. The statements and conclusions published by the author are the results of his own study and do not necessarily reflect the opinion of the Veterans Administration.     

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5 % of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 g/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

Deoxyribonucleic acid base composition of the yeastlike and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis

The base composition in moles percent guanine plus cytosine (%GC) of both nuclear and mitochondrial deoxyribonucleic acid (DNA) isolated from the yeastlike and mycelial phases of the dimorphic fungal pathogens Histoplasma capsulatum and Blastomyces dermatitidis was determined by techniques of thermal denaturation and CsCl buoyant density gradient equilibrium centrifugation. The mean observed values for GC content of nuclear DNA from H. capsulatum and B. dermatitidis were 47.3 and 48.2%, respectively. What is speculated to be mitochondrial DNA was found to be 34.0% for H. capsulatum and 34.3% for B. dermatitidis. Thermal denaturation curves for Blastomyces DNA indicated a bimodality in thermal denaturation profiles, thereby suggesting a significant mitochondrial DNA contamination. Mitochondrial DNA appeared to represent a smaller percentage of the total DNA prepared from Histoplasma, and was not observed consistently to affect%GC values as determined by thermal denaturation profiles. On the basis of the now known perfect stage of B. dermatitidis (Ajellomyces dermatitidis) as a member of the family Gymnoascaceae, the close approximation of%GC content of nuclear DNA of this fungal organism with that of H. capsulatum suggests possible phylogenetic relationship. It is suggested that the just reported, but as yet unclassified, perfect stage of H. capsulatum may be found to be phylogenetically a primitive form of the Gymnoascaceae.     

Chemical and immunological properties of galactomannans obtained from Histoplasma duboisii,Histoplasma capsulatum,Paracoccidioides brasiliensis and Blasomyces dermatitidis

Serologically active polysaccharide, galactomannan, was isolated from whole cells ofH. capsulatum, H. duboisii, P. brasiliensis andB. dermatitidis, and their chemical structure and immunological properties were described.     

Protoplasts of systemic dimorphic fungal pathogens: Histoplasma capsulatum and blastomyces dermatitidis

Summary Protoplasts have been released fromH. capsulatum in the mycelial and yeast phases and from the mycelial and incompletly converted yeast phase ofB. dermatitidis by the enzymatic action of snail digestive juice. There is great variation in the mode of protoplast formation not only between species but between the two morphological forms, particularly inH. capsulatumThese studies were supported in part by grants from the American Thoracic Socicty and the United States Public Health Service National Institute of Allergy and Infectious Diseases (1-R1-AI-7520-01).     

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum

The ultrastructural changes produced by ketoconazole on the yeast-phase ofH. capsulatum andP. brasiliensis were studied by means of scanning and transmission electron microscopy.The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure ofC. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole thanH. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 gmg/ml and in the latter at 1 g/ml.     

Antigenic Relationship Between American and African Isolates of Blastomyces dermatitidis as Determined by Immunofluorescence

The antigenic relationship between American and African isolates of Blastomyces dermatitidis was investigated through the use of the fluorescent antibody technique. Preliminary results suggest that the American and African isolates studies are not antigenically identical. The American isolates share antigens with the African ones; however, it appears that they also possess distinct antigens. Additional studies are necessary to confirm these findings and to draw any conclusion whether the African isolates represent a serotype(s) of B. dermatitidis or a distinct species.     

Rapid Identification of Histoplasma capsulatum Directly from Cultures by Multiplex PCR

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.     

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg⁺² optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.     

Utilization and cell-surface binding of hemin by Histoplasma capsulatum

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

Ultrastructural alterations of the mitochondrial ATPase in the calcium paradox as revealed by negative staining

We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P less than 0.05) decrease in the number of spheres on the mitochondrial membranes occurs during the calcium paradox (58.0 +/0 4.1/micrometer vs. control 80.5 +/- 6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5 +20 5.0 micrometer). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs.