Elizabeth J. Keath

Molecular cell biology and molecular genetics of Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic ascomycete which is capable of producing a broad spectrum of disease ranging from mild asymptomatic, pulmonary illness to severe, life-threatening systemic mycosis. Regulatory mechanisms that use temperature and other environmental cues are paramount to the successful adaptation of the organism as an effective intracellular pathogenic yeast. Although the biochemistry and phenomenology of reversible morphogenesis have been well examined in Histoplasma, the identification and functional characterization of genes and their products that are required for early establishment or maintenance of the parasitic yeast phase in intracellular host compartments have only recently been fruitful. Advances in the molecular biology of Histoplasma, including approaches to introduce telomeric plasmids, reporter fusion constructs, and gene disruption cassettes into the fungus are poised to solidify the pre-eminence of this fungus as a model system which can be applied to other dimorphic fungal pathogens that exhibit similar cellular and immunological complexities. This review centers on recent developments in the molecular cell biology and molecular genetics of Histoplasma capsulatum that provide important new avenues for examining the mold-to-yeast phase transition beyond the historical, binary view of dimorphism and the implications that these successful approaches may have on seminal issues in fungal pathogenesis.

Fibroblast lines expressing activated c-myc oncogenes are tumorigenic in nude mice and syngeneic animals

The influence of c-myc expression on fibroblast growth and morphology was investigated by transfection of c-myc genes linked to viral promoters. No foci were observed after transfection of either NIH/3T3 or Rat 2 cells. Cell lines containing activated c-myc genes were established using SV2-neo coselection and several growth parameters of the cells were studied. The cells showed a slight increase in refractility and formed colonies in soft agar with an efficiency of only 1%-2%. The c-myc-transfected cells grew well in 0.5% serum while the controls did not. The major difference in cell growth noted was that c-myc-transfected cells were tumorigenic when inoculated into nude mice or syngeneic rats. Analysis of RNA from the tumorigenic cells showed a level of c-myc expression from the transfected genes that was 2 to 6 fold higher than that from the endogenous gene. The level of c-myc RNA in the fibroblast tumors was similar to that found in mouse plasmacytomas. Expression of the endogenous c-myc gene was unaffected by the transfected genes for subconfluent cells in culture, but the gene was shut off in the nude mouse tumors. These results demonstrate that constitutive c-myc expression leads to tumorigenicity in immortalized cell lines.

Differences in histoplasmosis in patients with acquired immunodeficiency syndrome in the United States and Brazil

Demographic and clinical parameters among patients with acquired immunodeficiency syndrome and histoplasmosis in Brazil and United States were compared. The Brazilian isolates were typed by restriction-fragment length polymorphism analysis and were DNA fingerprinted by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). Skin lesions occurred in 66% of Brazilian case patients, compared with 1%-7% of US case patients. Of 21 treated case patients, 4 (19%) died, a rate similar to that of the US case patients (5%-13%). By nuclear gene typing, the Brazilian isolates were equally divided between South American classes 5 and 6, and RAPD-PCR showed 18 distinct genetic fingerprints in 20 isolates. Skin lesions are more common in infection with class 5 or 6 organisms than with class 2 Histoplasma capsulatum. The role of genetic differences in the organism as a cause for the clinical differences requires investigation.

Transcriptional activation of the translocated c-myc oncogene in mouse plasmacytomas: Similar RNA levels in tumor and proliferating normal cells

We examine the influence of the immunoglobulin locus on the expression of the translocated c-myc oncogene in mouse plasmacytomas. The level of c-myc RNA was 30- 35-fold greater in tumor cells than in normal, quiescent B cells. Mitogen stimulation of the lymphocytes with lipopolysaccharide induced a 15-fold increase in c-myc expression per cell to a level that was similar to that in the transcription of the translocated c-myc gene involved initiation from sequences in the first c-myc intron. Abundant RNA transcripts were also found from the noncoding strand of the c-myc intron in most tumor lines. S1 nuclease mapping was used to locate the intronic sequences that are used to initiate the tumor-specific c-myc RNAs. Six different initiation sites within the intron were mapped, none of which have the TATA sequence usually associated with eucaryotic RNA polymerase II promoters. The noncoding strand transcripts were also found to initiate in the c-myc intron. Transcription of the c-myc coding strand was independent of the position of the translocation breakpoint, even when the heavy chain switch and constant regions were deleted.

Pathogenic Differences between North American and Latin American Strains of Histoplasma capsulatum var. capsulatum in Experimentally Infected Mice

ABSTRACT Clinical differences in histoplasmosis between North America and Brazil prompted investigation of experimental infection with representative strains. Mortality was higher with Latin American strains, and lung pathology showed large necrotizing granuloma with prominent neutrophilic infiltration. Chronic disease was unique to the North American strain.

Gel Shift Assay of Nuclear Extracts from Histoplasma capsulatum Demonstrates the Presence of Several DNA Binding Proteins

ABSTRACT A gel shift assay was optimized to detect several general DNA binding proteins from Histoplasma capsulatum strain G217B. The electrophoretic mobility shift assay (EMSA) technique also detected protein(s) recognizing a pyrimidine-rich motif found in several Histoplasma promoters. Establishment of EMSA conditions provides an important framework to evaluate regulation of homeostatic or phase-specific genes that may influence virulence in Histoplasma and other dimorphic fungal pathogens.