Influence of Oestradiol on Protein Expression and Methionine Utilization during Morphogenesis of Paracoccidioides brasiliensis

Abstract

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by onedimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17P-oestradiol. Using either protein staining or fluorography of [ SS]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 "C to 37 "C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 1 OW7 M-oestradiol altered observed profiles ; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeastspecific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding proteinligand complex.

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“…35 S]methionine uptake and incorporation by nontransforming mycelial cells at 25°C (26). To date, the induction of methionine uptake remains the only defined subcellular response to E 2 in these cells which our laboratories have found.…”

“…In contrast, in E 2 -treated cultures these bands were absent or delayed in appearance even after 120 h of transition. In addition, fewer total bands were present in the cytosols from E 2 -treated cultures after 120 h of transition than from the control (26).…”

“…Interestingly, there was an overall difference in the number of labeled bands observed in the profiles after 120 h of transition. [ 35 S]methionine incorporation, and presumably de novo protein synthesis, was reduced, with many fewer protein bands being observable for the E 2 -treated cultures, whereas the control cultures had continued de novo protein synthesis and exhibited a yeast protein profile (26). Overall, these results indicated that a switch to a utilization of exogenous methionine occurs within 24 h of a temperature increase to 37°C, that the synthesis of yeast proteins began 24 to 72 h before observable morphological changes occurred, and not only that the presence of E 2 blocked the form transition from mycelia to yeast but also that de novo protein synthesis was almost totally downregulated.…”

“…Furthermore, the distinctive protein profiles of the mycelial and yeast forms of P. brasiliensis are suggestive of differential protein expression based on temperature (26).…”

“…A comparison of only the mycelial and yeast protein profiles showed that 30 bands were distinctly specific to either the mycelial or yeast form: 12 bands for the mycelial form (ranging from approximately 30 kDa to 140 kDa) were not found for the yeast form, and 18 bands (ranging from approximately 22 kDa to 127 kDa) for the yeast form were not found for the mycelial form (26).…”