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Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum.

ABSTRACT: Histoplasma capsulatum is a pathogenic fungus that exists in two distinct forms. The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes. Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism. In this study, we show that the gene encoding CBP (CBP1) is transcriptionally regulated. To identify promoter sequences that are important for yeast phase-specific activity, we created a series of fusions between successively truncated CBP1 5' untranslated regulatory sequences and the Escherichia coli lacZ gene. The fusions were constructed on a telomeric shuttle plasmid that can replicate autonomously in the fungus. By assaying for beta-galactosidase activity from H. capsulatum transformants, we identified a 102-bp region that mediates promoter activation and yeast phase promoter activity. Base pair substitution analysis suggests that the sequences between 839 and 877 bp upstream of the start codon are the most important for this positive regulation.

SUBMITTER: Patel JB

PROVIDER: S-EPMC107091 | biostudies-literature | 1998 Apr

REPOSITORIES: biostudies-literature

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Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum.

Patel J B JB Batanghari J W JW Goldman W E WE

Journal of bacteriology 19980401 7

Histoplasma capsulatum is a pathogenic fungus that exists in two distinct forms. The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes. Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism. In this study, we show that the gene encod ...[more]

PMID: 9537376

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Project description:The dimorphic fungus Histoplasma capsulatum causes respiratory and systemic disease in mammalian hosts by expression of factors that enable survival within phagocytic cells of the immune system. Histoplasma's dimorphism is distinguished by growth either as avirulent mycelia or as pathogenic yeast. Geographically distinct strains of Histoplasma differ in their relative virulence in mammalian hosts and in production of and requirement for specific virulence factors. The close similarity in the genome sequences of these diverse strains suggests that phenotypic variations result from differences in gene expression rather than gene content. To provide insight into how the transcriptional program translates into morphological variation and the pathogenic lifestyle, we compared the transcriptional profile of the pathogenic yeast phase and the non-pathogenic mycelial phase of two clinical isolates of Histoplasma.To overcome inaccuracies in ab initio genome annotation of the Histoplasma genome, we used RNA-seq methodology to generate gene structure models based on experimental evidence. Quantitative analyses of the sequencing reads revealed 6% to 9% of genes are differentially regulated between the two phases. RNA-seq-based mRNA quantitation was strongly correlated with gene expression levels determined by quantitative RT-PCR. Comparison of the yeast-phase transcriptomes between strains showed 7.6% of all genes have lineage-specific expression differences including genes contributing, or potentially related, to pathogenesis. GFP-transcriptional fusions and their introduction into both strain backgrounds revealed that the difference in transcriptional activity of individual genes reflects both variations in the cis- and trans-acting factors between Histoplasma strains.Comparison of the yeast and mycelial transcriptomes highlights genes encoding virulence factors as well as those involved in protein glycosylation, alternative metabolism, lipid remodeling, and cell wall glycanases that may contribute to Histoplasma pathogenesis. These studies lay an essential foundation for understanding how gene expression variations contribute to the strain- and phase-specific virulence differences of Histoplasma.

Project description:Genes expressed in the parasitic yeast (Y) phase of the dimorphic fungal pathogen Histoplasma capsulatum which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To understand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5' regulatory regions of two Y phase-specific genes (yps-3 and yps 21:E-9) were PCR amplified as labelled probes to identify nuclear DNA binding proteins which may influence phase-specific gene transcription. Protein-DNA interactions were assessed by Southwestern blot analysis in which sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated protein extracts from Y and M phases of the virulent G217B strain of H. capsulatum were visualized by their capability for in situ binding to the labelled 517-bp (G217B yps-3) or the 395-bp (G217B yps 21:E-9) putative 5' regulatory regions. A 30-kDa nuclear protein unique to the M-phase extracts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, thermal-sensitive Downs strain of H. capsulatum lacked detectable p30 binding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B yps 21:E-9 probe or 512-bp HindIII-EcoRI-labelled Downs yps21:E-9). A decanucleotide motif, TCCTTTTTTT, was identified in the upstream regulatory regions of these yps genes, as well as in the putative alpha-tubulin promoter, and was conserved with 70 to 100% homology. This recognition sequence was sufficient for p30M binding with 32P-labelled ligated oligonucleotides when used in the Southwestern assay. These findings describe the first nuclear DNA binding factor identified in H. capsulatum which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in which a temperature-sensitive switch influences morphology and virulence.

Dataset Information

Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum.

Publications

Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum.

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