Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum.

Abstract

Recent investigations have confirmed the presence of one a-tubulin gene (TUB)) and one ,B-tubulin gene (TUB2) in the dimorphic fungus Histoplasma capsulatum. In the present study, Northern blot (RNA blot) analyses revealed multiple a-tubulin transcripts and a single i-tubulin transcript in the yeast and mycelial phases of the high-virulence 217B strain and low-virulence Downs strain. Si nuclease protection assays demonstrated one initiation start site and two major stop sites for the TUB) transcripts, suggesting that variations in 3' processing generate the a-tubulin messages of 2.5 and 2.0 kilobases. Dot blot hybridization experiments indicated that tubulin gene expression is developmentally regulated during the dimorphic phase transitions. a-and (-tubulin mRNAs increased six-to eightfold during the yeast-to-mycelium conversion and decreased two-to threefold during the reverse transition. These changes in tubulin mRNA content coincided with major morphological events associated with H. capsulatum development. Western blots (immunoblots) of H. capsulatum yeast-specific proteins resolved by two-dimensional gel electrophoresis demonstrated a single aand a single j-tubulin isoform. Multiple tubulin polypeptides expressed in mycelia are probably products of posttranslational modffications.Histoplasma capsulatum, a dimorphic fungal pathogen, grows in soil as multicellular filamentous mycelia and in the tissues of infected animals and humans as unicellular budding yeast cells. The mycelial and yeast phases may be maintained in vitro at 25 and 37°C, respectively. Conversion of mycelia to yeast cells is induced in the laboratory by shifting the temperature of incubation from 25 to 37°C. A temperature shift from 37 to 25°C will initiate the reverse transition.Dimorphism in H. capsulatum involves elaborate changes in cell morphology, and elucidation of the mechanism involved may provide insight into differentiation and development in higher organisms. Changes in cell shape in H. capsulatum are likely to require alterations in cytoskeletal components; the arrangement of microtubules at the cell surface, for example, may direct subsequent morphological events. Previous studies in this laboratory suggest that tubulin, the major constituent protein of microtubules, plays an active role in the H. capsulatum differentiation process. Methyl benzimadazole-2-yl carbamate (MBC), an agent which binds specifically to fungal tubulins, inhibits the dimorphic phase transitions (J. Medoff and G. Medoff, Abstr. Annu. Meet. Am. Soc. Cell Biol. 1982, 95:44a Harris, E. J. Keath, and J. Medoff, J. Gen. Microbiol., in press). Hybridization probes from the cloned a-tubulin gene (TUBI) and p-tubulin gene (TUB2) were used in the present study to examine tubulin-specific expression during morphogenesis. The results presented here reveal that tubulin mRNA production is developmentally regulated during the dimorphic phase transitions.

The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions.

“…We have also investigated the splicing activity of another intron-containing gene, the ,-tubulin gene, to determine whether the H. capsulatum hsp82 locus had evolved a heat shock-resistant structure or whether splicing itself is more resistant in this organism. The P-tubulin gene, which contains eight intervening sequences (12), is developmentally regulated, with increases in mRNA transcription of six-to eightfold during the yeast-to-mycelium phase transition and two-to threefold during the reverse transition (11). These increases are measurable 1 to 3 days after temperature shifts, while in the first 12 h of the mycelium-to-yeast phase transition there is a decrease in mRNA transcription (11).…”

“…My, mycelia. differences in the hybridization pattern were obtained with the H. capsulatum hsp7O and ,-tubulin genes when total DNAs prepared from the same two strains were analyzed (7,11).…”

“…The P-tubulin gene, which contains eight intervening sequences (12), is developmentally regulated, with increases in mRNA transcription of six-to eightfold during the yeast-to-mycelium phase transition and two-to threefold during the reverse transition (11). These increases are measurable 1 to 3 days after temperature shifts, while in the first 12 h of the mycelium-to-yeast phase transition there is a decrease in mRNA transcription (11). This decrease correlates to the general decline in RNA transcription due to the heat shock condition of the mycelium-to-yeast phase transition (7,12,21,22,35).…”

Characterization of Alpha and Beta Tubulin Genes in the Dimorphic Fungus Histoplasma capsulatum

“…The analysis of tubulin-specific mRNAs during H . capsulatum morphogenesis reveals that alpha and beta tubulin gene expression is induced coordinately in response to temperature shift (Harris et al, 1989). Coordinate gene regulation in this fungus may imply the presence of common regulatory features which direct the synchronous response.…”

Histoplasma capsulatum Molecular Genetics, Pathogenesis, and Responsiveness to Its Environment

“…Finally, a number of genes have shown differences in relative expression although not completely morphotype-specific expression. For instance, cdc2 (Di Lallo et al, 1994) and a calmodulin gene (el-Rady and Shearer, 1996) are expressed more highly in yeast, genes encoding both ␣ and ␤ tubulins are expressed more highly in mycelia (Harris et al, 1989), and the actin gene shows a transient decrease in expression during the myceliayeast transition (el-Rady and Shearer, 1997).…”