Inhibitory effects of organic sulfur compounds on Histoplasma capsulatum

Summary Eight classes of organic sulfur compounds, comprising 42 substances, were tested in vitro for activity against the yeast phase ofHistoplasma capsulatum Darling. Activity was found in the classes of thiols, thio acids, disulfides, and thiolsulfonates. The classes of sulfinates, sulfenamides, penicillamine analogs, and reaction products of thiols with aldehydes or ketones were unpromising. Tentative conclusions are drawn of structure-activity relations within classes as a guide to coupling of classes to form disulfides.This investigation was supported by Biomedical Science Support Grant, National Institutes of Health Grant FR-07089-02 to Vanderbilt University (Ilda McVeigh) and by PHS Research Grant AM-11685 from the National Institute of Arthritis and Metabolic Diseases (Lamar Field).     

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.     

Inhibition of growth of Histoplasma capsulatum by lymphokine-stimulated macrophages

Peritoneal cells from mice immunized by sublethal infection inhibit the intracellular growth of Histoplasma capsulatum in vitro. Lymphokines generated in cultures of immune splenocytes stimulated with Histoplasma antigen activate normal macrophages to inhibit the intracellular growth of the fungus. Such lymphokines may inhibit the intracellular growth of H. capsulatum by 60 to 80% but have no direct effect on the viability of the fungus extracellularly. The lymphokine preparations have high interferon activity that is heat stable and acid labile. The cells in the spleen that are responsible for lymphokine production are T lymphocytes, but the help of other cells such as macrophages is essential for maximal production.     

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.     

Environmental factors and growth of Histoplasma capsulatum in soil

Summary Environmental factors influencing the growth ofHistoplasma capsulatum in soil have been studied. The role of temperature and moisture in the growth of the fungus was found to be critical. The fungus can tolerate very low temperatures, if the soil moisture content is high, but cannot withstand temperatures of 40° C or above for an extended period.Dry, sterile chicken manure and an extract of unsterile chicken manure showed an inhibitory effect on the growth of the fungus. However, the relationship between bird manure andH. capsulatum has not been satisfactorily clarified.Morphological studies ofH. capsulatum in soil showed that the fungus grows within the upper two inches of the soil and a majority of sporulation occurs within the upper one-half inch of soil. Morphological studies of the fungus, in the presence of chicken manure and chicken manure extract, showed an increased number of macroconidia and microconidia and decreased mycelial production.The growth ofH. capsulatum in soil is markedly affected by soil pH above 10 and below 5. No growth was observed on soil cultures outside these values. There was no definite pH range within these values in which the fungus grew more abundantly.     

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.     

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.     

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.     

Selenocystine-resistant mutants of Histoplasma capsulatum

Cysteine metabolism has been thought to be important to the phenomenon of dimorphism inHistoplasma capsulatum. We sought mutants with genetic blocks in the metabolism of cysteine by selection of colonies resistant to the toxic analogue, selenocystine. The 22 resistant strains thus obtained were all deficient in uptake of cystine from the surrounding medium but were normally able to convert from mycelium to yeast and back again. Furthermore, they had normal quantities of NADH-dependent cystine reductase when this enzyme was measured. We conclude that mutants defective in cystine uptake can be readily obtained by selection of colonies resistant to selenocystine, and that a lesion in cystine-uptake does not appear to affect the phenomenon of dimorphism in this organism.Preliminary reports of this work were presented at the Second International Congress of Mycology, Tampa, 1977 and at the first International Conference on Histoplasmosis, Atlanta, 1978.