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.     

Preparation and standardization of antigens of Histoplasma capsulatum and Coccidioides immitis

Methods have been developed for the separation and purification of various antigenically active cellular components ofH. capsulatum (34) andC. immitis (35), and chemical procedures have been employed in the characterization of these antigens. The concluding remarks illustrate the current trends in immunological studies ofH. capsulatum andC. immitis. Hopefully, these approaches will provide knowledge and insight into the basic biological properties ofH. capsulatum andC. immitis and should, in the future, culminate in the physicochemical characterization of their important antigens.     

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.     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Relationship between age and cellular suppressive activity in resistance to Histoplasma capsulatum infection

One-month-old and 1-year-old male BALB/c mice showed a lower resistance than 4.5-month-old mice to Histoplasma capsulatum infection. 4.5-month-old mice successfully resolved the infection when challenged with either a LD50 or LD100 for 1-month-old mice. A critical clinical course of experimental histoplasmosis was observed in 4.5-month-old syngeneic mice when spleen cells from 1-month-old BALB/c mice were transferred to them. Irradiated recipient mice, into which bone marrow and spleen cells were transferred, died when infected with the LD100 for 1-month-old mice. The same occurred with 4.5-month-old non-irradiated infected mice which received only spleen cells and with 1-month-old mice which were used as a control of infection. However, infected and non-transferred 4.5-month-old mice survived this dose. Thus, the adoptive transference of spleen cells from 1-month-old mice to 4.5-month-old mice suppressed the resistance of these adult mice to infection. Apparently, the transference of the suppressive state requires the presence of two cell populations, a non-adherent and an adherent and radioresistant cell present in the spleen of male 1-month-old mice.     

Interactions between clay minerals and siderophores affect the respiration of Histoplasma capsulatum.

The reduction in the respiration of Histoplasma capsulatum in broth culture caused by montmorillonite appeared to be the result, in part, of the interference by the clay with the iron nutrition of the fungus. This interference was apparently the result of the adsorption by the clay of the iron-transporting siderophore (deferricoprogen B) produced by the fungus, as the reduction in respiration was partially alleviated by the addition of foreign siderophores. Neither kaolinite nor attapulgite (palygorskite) appeared to adsorb significant amounts of the siderophores, probably because of the low cation exchange capacity and specific surface area of kaolinite and the inaccessibility of adsorption sites in the fibrous attapulgite. These observations, in addition to the adhesion of montmorillonite to the hyphae, suggest mechanisms that may explain the discrete geographic distribution of this fungus, which is pathogenic to humans and which has been isolated essentially only from soils that do not contain montmorillonite.     

Respiration in the yeast and mycelial phases of Histoplasma capsulatum.

Respiration in the yeast and mycelial phases of Histoplasma capsulatum proceeds via a cytochrome system and an alternate oxidase, both present constitutively. The mycelial cytochrome system is distinguished by an additional partial shunt around the antimycin-sensitive site.     

Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.     

Amphotericin B-induced changes in K+ content, viability, and ultrastructure of yeast-phase Histoplasma capsulatum.

Yeast-phase cells of Histoplasma capsulatum were challenged with amphotericin B, and membrane perturbation was monitored by K+ efflux. Suspensions of washed cells readily absorbed about 1.12 microgram of amphotericin B per mg (dry weight) and further nonspecific sites were also apparent. The dose-response curve for initial rate of K+ efflux was sigmoidal within the range 0.1 to 1.0 microgram of amphotericin B per ml. A fungistatic concentration of amphotericin B (0.3 microgram/ml) evoked an efflux of 85 to 90% K+ from the cells within 15 min, but cell viability decreased only 13% (yeast phase) or 33% (transformed to mycelial units). Ultrastructural changes in treated cells were detected within 5 min, and the hallmark was expansion of vacuoles during the 1-h monitoring period. In contradistinction to a previous report, the appearance of the protoplasmic membrane was not altered by fungistatic concentration. When treated cells were returned to a fresh growth medium, there was a pronounced lag (20 h). During this apparent recovery phase, the large vacuoles fragmented and returned to normal size. It is proposed that vacuoles of H. capsulatum act as a spatial buffer of considerable survival value to stressed cells.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis.

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.     

Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of Histoplasma capsulatum.

The respiration of three phenotypes of Histoplasma capsulatum, the causal agent of histoplasmosis in humans, was markedly reduced by low concentrations of montmorillonite but was reduced less by even higher concentrations of kaolinite or attapulgite (palygorskite). The reduction in respiration followed a pattern that suggested saturation-type kinetics: an initial sharp reduction that occurred with low concentrations of clay (0.01 to 0.5% [wt/vol]), followed by a more gradual reduction with higher concentrations (1 to 8%). Increases in viscosity (which could impair the movement of O2) caused by the clays were not responsible for the reduction in respiration, and the clays did not interfere with the availability of nutrients. Scanning electron microscopy after extensive washing showed that the clay particles were tightly bound to the hyphae, suggesting that the clays reduced the rate of respiration of H. capsulatum by adhering to the mycelial surface and, thereby, interfered with the movement of nutrients, metabolites, and gases across the mycelial wall.     

Monocyte adherence to Paracoccidioides brasiliensis,zymosan-C3b and erythrocyte-hemolysin in patients with paracoccidioidomycosis

Independent and dependent (C3b/Fc receptors) opsonic adherence ability of monocytes from thirty-three patients with acute or chronic paracoccidioidomycosis and from 13 healthy individuals were studied in the presence of Paracoccidioides brasiliensis (Pb), Paracoccidioides brasiliensis opsonized by patient's serum (PbPS) or normal serum (PbNS), zymosan opsonized by fresh sera from healthy donors (ZyNS) and erythrocytes opsonized by hemolysin (EA). Statistically significant differences concerning the percentage of adhered monocytes to PbPS (number of adhered monocytes/total number of monocytes) were detected between control and chronic (active and inactive) groups. Significant differences in relationship to the mean number of PbPS (number of fungi in monocytes/total number of monocytes) were also observed between control and chronic active mycosis. Present data suggest that patients with chronic disease have more ability in the first step of phagocytic activity, considered as the main effector mechanism to control the dissemination and severity of paracoccidiodomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.