Phylogeography of the fungal pathogen Histoplasma capsulatum

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein‐coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein‐coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.     

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N₂O using NADH as the direct reductant. The K_M for NO was determined to be 20 μM and the k_cat to be 5000 min⁻¹. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.     

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. capsulatum These 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).     

Detection and Identification of Diagnostic Histoplasma capsulatum Precipitates by Counterelectrophoresis

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.     

Molecular mycology: a genetic toolbox for Histoplasma capsulatum

Research in medical mycology has traditionally been a mix of exciting biology and frustrating genetics, although the excitement has been steadily increasing as genetic obstacles have been successfully overcome. Now, a variety of fungal pathogens can be studied using molecular techniques derived from classical bacterial and yeast genetics, but with selective and strategic adaptations. Histoplasma capsulatum is the best-studied of the primary pathogens known as 'dimorphic' fungi, and tailored molecular genetic strategies are beginning to reveal a repertoire of genes and gene products intimately associated with pathogenesis.     

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.     

Isolation of Histoplasma capsulatum from a texas cave

Summary Sixty-three soil samples collected from caves in Texas and the Republic of Mexico were studied to determine the incidence of pathogenic fungi by the use of the flotation technique in mice.Histoplasma capsulatum was isolated from the Kickapoo cave in Texas, and there was some histopathological evidence of its presence in the soil of Devil' cave (Cueva del Diablo) in Mexico. In the latter case, an incidence of human contact with the cave which resulted in an illness is presented.     

Cysteine biosynthesis in a fungus, Histoplasma capsulatum.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.     

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP–GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.     

Improved technique for the reversion of Histoplasma capsulatum in tissue culture

Summary Reversion ofHistoplasma capsulatum mycelia to the parasitic yeast phase was accomplished in stationary cultured HeLa cells. Fresh guinea pig serum in either Hely or Medium 199 gave excellent conversion. Horse, human, calf, and chicken serum permitted conversion, but to a lesser extent as measured by subsequent growth on blood agar. No apparent differences were found with respect to conversion between 2, 4, and 8-week old mycelial inoculum.Work reported in this paper was supported in part by Grant 2 T1 AI 123 of the National Institutes of Health, U.S. Public Health Service.     

Histoplasma capsulatum in the environment of sporadic histoplasmosis cases

Conclusions 1. Acute pulmonary histoplasmosis in adults is demonstrated to be associated with exogenous sources of infection in one-half of a series of cases. 2. No exogenous sources of infection were found in chronic pulmonary histoplasmosis in adults by methods comparable to those used in acute pulmonary histoplasmosis. 3. The significance of these findings for the pathogenesis of the three clinical forms of histoplasmosis is discussed. 4. In acute pulmonary histoplasmosis in adults, the isolation ofH. capsulatum from the environment at sites of exposure aided in specific diagnosis.Two acute pulmonary histoplasmosis cases were reported previously (2–3).     

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

BackgroundCurrent methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.ObjectivesThus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).MethodsThree DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.ResultsThe DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood &; Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.ConclusionsIn our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.