Identification and characterization of a phase-specific, nuclear DNA binding protein from the dimorphic pathogenic fungus Histoplasma capsulatum

Abstract

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.

FIG. 1

Sequences of the putative 5′ regulatory regions of the genes utilized as probes in Southwestern analysis. The flanking (5′ and 3′) primers used for probe amplification are indicated by dashed lines. (A) The previously described yeast phase-specific gene, yps-3; (B) yps 21:E-9; (C) the limited 5′ regulatory region of the α-tubulin gene (TUB1). Both yps genes are expressed in the yeast phase of the virulent strain G217B, but they are both silent in the yeast and mycelial phases of the attenuated Downs strain. α-Tubulin is expressed in both phases of the Downs and G217B strains, with fivefold-higher transcript levels detected in the mycelial phase (9). The transcriptional start sites, determined by S1 mapping of G217B poly(A)⁺ RNA, for each yps gene is shown (underlined), as well as the putative translational start sites (START) for all three sequences.

FIG. 2

Southwestern blot analysis of extracts prepared from the yeast and mycelial phases of H. capsulatum strains. (A) Nuclear (Nucl) and cytoplasmic (Cyto) extracts (40 μg) prepared from the yeast and mycelial phases of the Downs and G217B strains were electrophoresed on SDS–10% polyacrylamide gels, fixed, and stained with Coomassie brilliant blue. (B) A second gel, electrophoresed in parallel under the same conditions, was transferred by electroblotting, blocked, and treated with 10⁵ cpm of the PCR-amplified target probe (395 bp of the 5′ regulatory region of yps 21:E-9 in G217B)/ml for 2 h. The blot was washed and autoradiographed to demonstrate a protein (designated p30M) of an apparent molecular mass of 30 kDa from the mycelial phase nuclear extracts of G217B which interacts with the ³²P-labelled DNA target in situ.

FIG. 3

Southwestern blot analysis of p30M binding with ³²P-labelled target probes from various sources. Nuclear extract (40 μg) prepared from the yeast (Y) and mycelial (M) phases of the G217B and Downs strains of H. capsulatum were electrophoresed on SDS–10% polyacrylamide gels, transferred, and allowed to interact with the indicated DNA target probes. All probes were prepared by PCR amplification, except for the Downs yps 21:E-9 probe, which required end labelling of the 512-bp HindIII-EcoRI fragment at the HindIII site.

FIG. 4

Localization of DNA binding activity to a 150-bp DdeI fragment in the upstream regulatory region of yps 21:E-9. Nuclear (Nucl) and cytoplasmic (Cyto) extracts (40 μg) from the mycelial phases of G217B and Downs were evaluated by Southwestern analysis with ³²P-labelled targets derived from the 395-bp PCR-amplified yps 21:E-9 regulatory region from G217B. The amplified probe was digested with DdeI to liberate labelled fragments of 109, 150, and 136 bp. As indicated in the diagram, the 135-bp fragment contains the ATG codon while the transcription start site mapped by S1 analysis is within the 150-bp fragment flanked by DdeI sites. The asterisk denotes the location of the single TCCTTTTTTT motif observed in this DNA ligand.

FIG. 5

Competition of the p30M interaction with the ³²P-labelled 395-bp G217B yps 21:E-9 target by increasing amounts of unlabelled decanucleotide. Extracts from the mycelial (M) and yeast (Y) phases of the Downs and G217B strains were electrophoresed, blotted, and then evaluated for DNA binding to labelled probe in the absence and presence of the cold, ligated oligonucleotide (oligo) probe. The cold DNA ligand represented concatemers of the TCCTTTTTTT sequence and its complement and were ligated to an average range of 150 to 350 bp, as assessed by agarose gel electrophoresis.

FIG. 6

Southwestern analysis of nuclear extracts prepared from mycelial phase-to-yeast phase transforming cultures of G217B. Mycelial cells of strain G217B were induced to transform to the yeast phase at 37°C, and nuclear extracts were obtained from the terminal phases as well as 1 day, 3 days, 8 days, and 11 days following the shift-up in temperature. Proteins were electrophoresed in duplicate on SDS–10% polyacrylamide gels; half of the gel was stained to verify protein loading, and the remainder was transferred and evaluated for DNA binding. The results shown are from separate Southwestern blots with the 335-bp regulatory region of TUB1 (the α-tubulin gene) amplified from G217B and the 5′ regulatory region of yps 21:E-9 amplified from G217B.