Effect of thimerosal on the whole yeast phase antigen of Histoplasma capsulatum

Summary Thimerosal (merthiolate) and formalin treated whole-cell yeast phase antigens ofHistoplasma capsulatum were prepared and their reactivities with sera from cases of histoplasmosis, blastomycosis and coccidioidomycosis were compared. Thimerosal treated antigens often gave complement fixation titers with heterologous sera 2 to 8 fold lower than the titers obtained with formalin treated antigens. However, with certain anti-histoplasmosis sera, thimerosal killed antigens had less reactivity with homologous antisera also. In virtually all cases an equal or higher specificity ratio was obtained with thimerosal killed antigens. The effects of thimerosal and formalin were independent, indicating different sites of reactivity of these reagents. Uptake of thimerosal at several concentrations suggested two types of reactions with live yeast phase cells. Analyses of the cellular fractions for thimerosal showed it was present only in the soluble fractions from which it was readily removed by dialysis. Cellular fractions killed with thimerosal retained several of the same physical and antigenic characteristics of those fractions isolated from frozen and thawed cells.     

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.     

Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum

We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.     

Inhibition of isolated yeast and mycelial phase RNA polymerases of Histoplasma capsulatum by rifamycin derivatives

Various derivatives of rifamycin were shown to inhibit the RNA polymerases of the yeast and mycelial phases of Histoplasma capsulatum. The relative potency of each of the derivatives against the isolated polymerases was the same as the potency of each against the viable organism. RNA polymerase PC III from the yeast phase was more susceptible to the rifamycin derivatives than yeast phase polymerases PC I and PC II and the biggest differences in susceptibility were seen with the derivative AF/ABDP (2,6-dimethyl-4-benzyl-4-demethyl-rifamycin). The susceptibility pattern of the mycelial polymerase activity was identical to the yeast polymerase PC III.     

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.     

Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.     

Antibiotic sensitivity of the yeast and mycelial phases of Histoplasma capsulatum.

Cycloheximide and chloramphenicol in combination have a greater effect on yeast cells of Histoplasma capsulatum than on the mycelial phase of this fungus. In contrast, clotrimazole was found to be more effective against mycelia. Miconazole produced a pronounced effect against both phases wheras tolnaftate was only slightly active. Sulfadiazine and griseofulvin were completely inactive against both phases. The differential sensitivities of the 2 phases of H. capsulatum to various antibiotics can be useful in studying the transition of the dimorphic fungus from 1 phase to the other.     

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.     

Effects of various compounds on growth of yeast cells of Histoplasma capsulatum

Summary A number of compounds were screened for their effects on growth of the yeast cells ofHistoplasma capsulatum. Included were penicillin and related compounds, sulfhydryl inhibitors, various organic sulfur compounds recently synthesized for the first time, and compounds structurally related to the required metabolites, thiamine and cystine or cysteine. Cephalothin was the only one of the penicillin related compounds which inhibited growth. This occurred only when a high concentration (8.3 × 10^–4 M) was used. Of the analogues of cystine tested, allylglycine had the greatest inhibitory effect on growth of the yeast cells in the synthetic medium, but it failed to inhibit growth in a complex medium containing peptones and plasma. Among the sulfhydryl inhibitors, the maleimides were the most effective, producing complete inhibition of growth in the peptone medium at 10µg/ml or less. At subinhibitory concentrations the cultures tended to become mycelial. The action of the maleimides was reversed by cystine over a range of concentrations. At low concentrations, some of the disulfide derivatives of thiamine stimulated growth equally as well as thiamine, but at concentrations of 100 to 150µg/ml, they completely inhibited growth. On the basis of results obtained to date, three classes of the new organic sulfur compounds being tested offer promise as sources of potentially useful chemotherapeutic agents. These classes, which differ widely in structure, are as follows: the benzyl decylaminoethyl disulfides, the acyl disulfides, and the trithiopercarbamates.This investigation was supported by Public Health Service Research Grant AI-03524 from the National Institute of Allergy and Infectious Diseases.     

Carbohydrate structures of three novel phosphoinositol-containing sphingolipids from the yeast Histoplasma capsulatum

From the yeast phase of the human pathogen Histoplasma capsulatum, three novel glycolipids were isolated, shown to react with sera from histoplasmosis patients, and partially characterized: compound V, ceramide-P-inositol-[mannose2]; compound VI, ceramide-P-inositol-[mannose2, galactose]; compound VIII, an isomer of compound VI [Barr, K., & Lester, R.L. (1984) Biochemistry (preceding paper in this issue)]. Ammonolysis of these lipids has yielded all the carbohydrate (oligosaccharides V, VI, and VIII) as novel, intact oligosaccharides suitable for characterization. Anomeric configurations were determined by specific glycosidase digestion and by the stability of peracetylated saccharides to CrO3 oxidation. Linkages were established by methylation analysis. These experiments yielded the following structural assignments: (formula; see text) The occurrence of galactofuranose is novel for glycosphingolipids, and it is noteworthy that compound VI is immunoreactive.