Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction

Abstract

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

FIG. 1.

Response to farnesol by C. albicans under conditions that promote GTF and hyphal growth. SC5314, CAI4, CAF2, rfg1/rfg (DU129), nrg1/nrg1 (DU152), tup1/tup1(BCa2-9), tup1/tup1 (BCa2-10), and TUP1/tup1 (BCa2-3) resting cells were inoculated into mGPP (pH 4.8) medium at 37°C in the presence or absence of 20 μM farnesol, and their cell morphologies were examined at 4 h. Scale bar = 10 μm.

FIG. 2.

TUP1 mRNA levels increased, while two Tup1-regulated genes, HWP1 and RBT1, were downregulated in the presence of farnesol (FOH). C. albicans SC5314 resting cells were inoculated into mGPP (pH 4.8) medium in the presence or absence of 20 μM farnesol and incubated at 37°C. Cells were then harvested at 0, 20, 40, 60, and 80 min postinoculation. Northern blots were prepared with total RNA from cells incubated in the presence or absence of farnesol. Shown is a phosphorimage of a representative Northern blot probed with radiolabeled TUP1 DNA (A), HWP1 DNA (B), and RBT1 DNA (C) and a plot of average mRNA levels from a minimum of three independent experiments. ACT1 mRNA levels were used as a loading control.

FIG. 3.

Tup1 protein levels are higher in the presence of farnesol. Total protein extracts were prepared from SC5314 and TUP1/tup1 (BCa2-3) resting cells inoculated into mGPP (pH 6.8) medium at 37°C in the presence or absence of 20 μM farnesol and incubated at 37°C for 60 min. The average change (fold) in Tup1 protein accumulation for farnesol-treated cells relative to that of untreated cells is shown. Act1 levels were used as a loading control.

FIG. 4.

Farnesol does not affect the expression of RFG1 or NRG1, which encode DNA binding proteins that function with Tup1, or EFG1, which encodes a transcription activator of hypha-specific genes. Quantitative Northern blotting analysis was used to measure the TUP1, NRG1, RFG1, and EFG1 mRNA levels in SC5314 at 60 min after the inoculation of resting cells under conditions that promote GTF in the presence and absence of 20 μM farnesol. The results are averages of three independent experiments.

FIG. 5.

Overproduction of farnesol by the tup1/tup1 mutant inhibits SC5314 filamentation. Resting cells were grown at 37°C for 24 h on yeast-peptone-dextrose agar plates to allow for farnesol accumulation in the agar (horizontal streak, C. albicans strain SC5314 [B and C]; or tup1/tup1, BCa2-10 [A and D]). Subsequently, either SC5314 (A and B) or tup1/tup1 (C and D) resting cells were plated (vertical streak) and incubated at 37°C for an additional 24 h. The areas above the two arrows (A and B, left panels) are zones of filament inhibition (as evident by smooth morphology) resulting from the farnesol produced by the horizontally streaked strains. Filamentation gives the wrinkled colony morphology seen below the arrows. The pictures in the two white boxes have been magnified ×2.5 so that the colony morphology can be seen more clearly (A and B, center panels). Micrographs of individual cells from the two bracketed regions are shown in the right panels (A and B; scale bar = 10 μm). The cells from the smooth regions are mainly yeast, while there is a much larger proportion of filamentous cells in the wrinkled region.