Alcohols inhibit translation to regulate morphogenesis in C. albicans

Abstract

Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans.

Keywords: Candida albicans; Eukaryotic initiation factor 2B (eIF2B); Filamentous growth; Protein synthesis.

Fig. 1

Concentration dependent inhibition of growth and protein synthesis by alcohols does not rely on GCN2. (A) Figure shows plots of growth rates at various concentrations of ethanol, butanol and isoamyl alcohol (IAA) as indicated. Wild type CAI4 strain and the mutant gcn2Δ strain were grown to OD₆₀₀ of 0.1, then treated and the OD₆₀₀ was taken hourly for 6 h to generate the growth rates plotted. (B) A bar chart depicting the fold inhibition in the rate of protein synthesis at the indicated concentrations of alcohol for wild-type and gcn2Δ cells. Translation rates were determined by measuring using ³⁵S methionine incorporation over a 10 min period. Each treatment was analysed in triplicate; error bars are ±SEM.

Fig. 2

Alcohols cause morphological differentiation in C. albicans. (A) Serial dilutions of overnight cultures of the wild type CAI4 strain were spread onto YPD agar plates containing 0.5% butanol, 2% ethanol, 0.5% IAA or 10% serum. Representative microcolonies were photographed (10× magnification) after 1 and 2 days of growth as indicated. (B) Images of cells from cultures that were grown at 37 °C in the indicated media for 3 h. (C) Cells from the analysis in B were scored for yeast, hyphal and pseudohyphal growth in a cell counting chamber. Percentages given are an average (±SEM) calculated from three biological replicates.

Fig. 3

Translation initiation is inhibited by alcohols in CAI4 and gcn2Δ strains. (A) Figure shows polysome analyses assessing the effect of alcohols on translation initiation in the CAI4 wild type and gcn2Δ strains of C. albicans. Strains were grown in YPD and the indicated concentrations of the alcohols were added for 15 min prior to extract preparation. Extracts were sedimented on 15–50% sucrose gradients and the absorbance at 254 nm was continuously measured to generate the traces depicted. The position of 40S, 60S, 80S and polysome peaks are labelled and the direction of sedimentation is also depicted. (B) Quantitation of the polysome: monosome ratio across the polysome profiles. Solid lines represent the wild type, dashed lines represent gcn2Δ strain.

Fig. 4

Alcohols do not induce eIF2α phosphorylation, but does induce GCN4 activation. (A and B) Luciferase assays from strains bearing either GCRE::Luc or GCN4::Luc reporters treated with the alcohols and concentrations indicated for 2 h. Values are plotted relative to untreated and represent a mean of three biological replicates. Error bars are ±SEM and all the alcohol dependent inductions are statistically significant (p < 0.05). (C) Western blots on extracts from wild type and gcn2Δ strains probed with antibodies to Tef1p and phospho-eIF2α. Strains were pretreated with 1 mM Cadmium then treated with various concentrations of alcohols (% v/v) for 15 min as indicated. Long and short exposures to film are indicated.

Fig. 5

Fusel alcohols and ethanol impede the movement of eIF2B bodies. (A) Images from time-lapse microscopy studies using an eIF2Bγ-GFP expressing C. albicans strain. The strains were incubated in media with 1% butanol, 0.5% IAA or 6% ethanol, or they were left untreated (UT) for 15 min as indicated. Each row contains two stills from a series of 25 images over a period of 2 min, as well as a merged image of all 25 stills, which serves to depict the total extent of 2B body movement. (B) Bar chart depicting the mean distance moved in μ over a 2-min period from 24 time-lapse experiments. Error bars, ±1 SEM; ^∗ p < 0.01.

Fig. 6

Separate alcohols affect morphological transcriptional regulator mutants. (A) Figure shows polysome analyses assessing the effect of alcohols on translation initiation in efg1Δ and cph1Δ mutant strains of C. albicans. Yeast strains were grown in YPD and various concentrations of alcohols were added as indicated for 15 min prior to extract preparation. Extracts were sedimented on 15–50% sucrose gradients and the absorbance at 254 nm was continuously measured. (B) Figure shows images of the mutant strains relative to wild type after treatment in liquid culture for 3 h with various alcohols, as indicated.

Fig. 7

Inhibition of protein synthesis by alcohols also impacts on filamentation in C. albicans. Figure shows micro-colonies formed after exponentially grown cells were plated on solid media containing the indicated supplements: (A) cycloheximide, (B) serum or ethanol, and (C) butanol, then grown for 1 day.