Uptake of Cystine by the Yeast Phase of Histoplasma capsulatum

This report deals with factors affecting the uptake of cystine by the yeast phase of Histoplasma capsulatum. The kinetics of uptake showed a saturation at 70 muM and an average K(m) value of 3 x 10(-5)m. The optimal pH and temperature for transport of cystine were 6.5 and 37 C, respectively. The energy of activation was 14.1 kcal/mole, and the temperature coefficient value was 2.1. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of incorporation of the amino acid by cells preincubated with either 2,4-dinitrophenol or sodium azide. Although uptake was not inhibited by any single amino acid, a combination of amino acids did cause a decrease in uptake. Thus, the data show that the uptake of cystine by yeast cells of H. capsulatum has the characteristics of a system of transport that requires the expenditure of energy by the cells.     

Immunogenicity of Ribosomal Preparations from Yeast Cells of Histoplasma capsulatum

Protective immunity was elicited by immunization of mice with ribosomal preparations from yeast cells of Histoplasma capsulatum. Ribosomes from disrupted cells were isolated by differential centrifugation using sodium dodecyl sulfate. These preparations contained 55% protein and 45% ribonucleic acid and sedimented as a single peak with a sedimentation coefficient of 77S on sucrose density gradient analysis. Mice immunized subcutaneously with ribosomes, with or without adjuvant, were challenged intravenously with 8 × 10⁶ yeast cells of H. capsulatum. Significant protection was induced by ribosomes and was greatly enhanced by adjuvants. Protection measured by 30-day survival compared favorably with the immunoprotection assessed by absence of lung lesions and negative spleen cultures. Treatment of ribosomes with ribonuclease before immunization reduced protection by 85%, whereas trypsin and Pronase reduced the protection by 50 to 55%. These findings indicate that both intact ribosomal ribonucleic acid and protein are necessary for maximal immunogenicity of Histoplasma ribosomes.     

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.     

Histoplasma capsulatum sinusitis.

Sinusitis is commonly reported in patients with AIDS. In addition to the usual bacterial pathogens isolated from immunocompetent patients, sinusitis in patients with AIDS may be caused by a variety of unusual bacteria, viruses, fungi, parasites, and mycobacteria. Histoplasma capsulatum has not typically been associated with sinusitis in either group of patients. We report a case of sinusitis caused by H. capsulatum in a patient with AIDS.     

Disseminated infection by Histoplasma capsulatum with AIDS

Histoplasmosis is an illness which occurs very rarely in Europe and it is especially rare in Germany. A generalised infection with Histoplasma capsulatum, a systemic mycosis of the mononuclear phagocyte system (MPS), occurs only in individuals with weakened immune systems. Within the framework of diagnostics, a pathologist can be confronted with histoplasmosis since there has been an increase in travel to and from endemic regions, as well as an increase in the number of diseases of the immune system. The presented case reports the histological intravital and post-mortem diagnostics of disseminated histoplasmosis in existing HIV-infection in the stage of manifest AIDS.     

Hydroxamate siderophores of Histoplasma capsulatum

The zoopathogenic fungus Histoplasma capsulatum, like other eukaryotic aerobic microorganisms, requires iron for growth. Under conditions of low iron availability, the fungus secretes hydroxamates that function as siderophores (iron chelators). The experiments to be reported were designed to gather further information on the hydroxamate siderophores of H. capsulatum. The fungus was grown in a synthetic medium deferrated with the cationic exchange resin Chelex 100. Siderophores were detected after 4 days of incubation at 37 degrees C in media containing 0.3 to 1.0 microM iron. The secretion was suppressed by 10 microM iron. The hydroxamates were purified by reverse-phase and size-exclusion chromatography. On the basis of ions observed during electrospray mass spectroscopy, five hydroxamate siderophores were tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was generated. This reagent cross-reacted with coprogen B and fusarinine. Thus, the antibody detects hydroxamates in all three families of siderophores excreted by H. capsulatum.     

A serendipitous isolate of Histoplasma capsulatum

This is a report of an unexpected laboratory diagnosis of Histoplasma capsulatum. The fungus was isolated from an acute cellulitic lesion on the forearm of an elderly male patient with a functioning renal transplant. The patient resides within the environs of Brisbane and has not travelled outside Australia. We consider the isolation of H. capsulatum from a rare site in a patient resident in a non-endemic area indicative of a latent opportunistic infection in an immunocompromised patient.     

An aberrant variant of Histoplasma capsulatum var. capsulatum.

We studied an aberrant culture of Histoplasma capsulatum var. capsulatum isolated from synovial fluid collected from the right elbow of a patient from Kansas. Colonies on Sabouraud glucose agar and other routine mycological media were glabrous to soft, moist, heaped, deeply folded or convoluted, and orange-brown with a white, irregular margin. Microscopically, hyphae were hyaline, septate, and branched and remained totally devoid of conidiation over a period of 2 years on all mycological media. Conversion to the yeast form was achieved on Pine's medium at 37 degrees C. Colonies at early stages of growth were smooth, moist, pasty, shiny, and orange-brown but soon became wrinkled and slightly raised and produced oval, thin-walled cells measuring 2 to 3 by to 4.5 microns which multiplied by polar budding. The identity of the isolate was further confirmed by utilizing the Accuprobe DNA and the exoantigen test for H. capsulatum var. capsulatum.     

Immunological studies on Histoplasma capsulatum.

Alveolar macrophages freshly harvested from normal and immunized rabbits were parasitized with yeast cells and protoplasts of Histoplasma capsulatum. Macrophages obtained from either normal or sensitized rabbits failed to phagocytize protoplasts, whereas, the yeast cells were actively ingested. There was no detectable intracellular killing by macrophages. A serological similarity was found between the whole yeast cell, the purified isolated cell wall, and the protoplasts of the fungus. Aprecipitin test of the protoplasts of the fungus gave a postive band, whereas immunodiffusion in agar was negative. Addition of immune sera activated phagocytosis, the immune sera against cell walls being the most active.     

Characterization and Evaluation of a Soluble Antigen Complex Prepared from the Yeast Phase of Histoplasma capsulatum

Soluble yeast-phase (YPS) antigen preparations (Reeves et al., 1972) were obtained from three strains of Histoplasma capsulatum. These were analyzed by agar-gel diffusion and complement fixation tests with human sera from known cases of histoplasmosis. Two of the preparations contained the H and M antigens normally found in histoplasmin, whereas the third preparation contained the H but not the M antigen. In addition, a group of unknown antigens, designated as the Y (yeast) antigens, were demonstrated in all three YPS preparations. On the basis of Sephadex gel filtration data, the molecular weight of the YPS M antigen was estimated to range from 117,000 to greater than 200,000; that of the YPS H antigen was estimated to range from about 60,000 to greater than 200,000; and that of the Y antigens was estimated to range from less than 10,000 to 100,000. Complement fixation tests with human homologous and heterologous sera showed that the Y antigens were specific for H. capsulatum; Y antigens were not detected in the single lot of histoplasmin used in this study. When 60 human histoplasmosis sera and serum samples from nine animals experimentally infected with H. capsulatum were analyzed, it was found that antibodies to the Y antigens occurred with about the same frequency as antibodies to the H antigen but with less frequency than that of antibodies to the M antigen. When used in rabbits as immunogens for the preparation of specific antisera to H. capsulatum, the components of the YPS preparations caused the formation of numerous cross-reacting antibodies. The data from this study show that the value of the YPS preparations for the serological diagnosis of histoplasmosis rests on the specificity of the H, M, and Y antigens and on the fact that the primary production of antibodies is restricted to these antigens in the course of natural infections. The YPS preparations were found to be stable for a period of at least 11 months under a variety of storage conditions and temperatures. Data obtained with various killing agents and metabolic inhibitors suggest an improved method for preparing the YPS antigens by using a suitable strain and killing the cells with iodoacetate.     

Identification of Histoplasma capsulatum from culture extracts by real-time PCR

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.     

Activation of the alternative complement pathway by Histoplasma capsulatum.

The present study was performed to determine whether Histoplasma capsulatum (yeast phase) is able to activate the alternative complement pathway. H. capsulatum yeasts were shown to consume C3 in C4-deficient guinea pig serum. Immunoelectrophoretic conversion of alternative pathway component factor B confirmed that C3 consumption was mediated by activation of the alternative pathway. These results demonstrate that H. capsulatum is able to activate the alternative complement pathway.     

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.     

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.     

Histoplasma capsulatum infection in nude mice.

Congenitally athymic nude (nu/nu) mice, when injected intraperitoneally with Histoplasma capsulatum, developed a rapidly fatal disseminated infection characterized by heavy parasitization of reticuloendothelial tissues. In contrast, their heterozygous (nu/X) littermates, which possessed a functioning thymus, developed only a low-grade infection which was apparently self-limited and rarely fatal. Transplantation of thymic tissue into nu/nu mice diminished greatly the severity of infection and reduced mortality by about 50%. These studies emphasize the importance of cell-mediated immunity in host defense against histoplasmosis and suggest that the nude mouse may be a valuable model for the study of this chronic intracellular infection.     

Fosmid-based physical mapping of the Histoplasma capsulatum genome

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.