N-acetylglucosamine induces white to opaque switching, a mating prerequisite in Candida albicans

Abstract

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO(2), equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans.

Figure 1. GlcNAc induces switching from white to opaque in a and α cells of Candida albicans.

A. The experimental protocol for assessing GlcNAC induction. Cells are first grown in liquid glucose medium at 25°C and then plated on glucose or GlcNAc agar at 25°C and in select cases at 37°C. B. Growth kinetics of a and α cells in liquid glucose medium at 25°C. C. Switching frequencies (Switch. Freq.) on glucose or GlcNAc agar of cells grown at 25°C in liquid glucose medium for 1, 2, 5 and 10 days. The colony number assessed for phenotype varied between 370 and 700 under the varied conditions. D. A graph of the switching frequencies from panel C. E. Examples of 3 day cultures on glucose or GlcNAc agar at 25°C. Switching frequencies were measured after 5 days on agar. No. Cols., number of colonies.

Figure 2. Ras1 plays a major role in GlcNAc induction.

A. Switching frequencies (Switch. Freq.) of wild type (WT) and ras1/ras1 cells grown in a glucose liquid medium at 25°C for 7 days and then plated on glucose agar and GlcNAc agar at 25°C. The colony numbers assessed for phenotype varied between 600 and 1,000 under varied conditions. B. Colonies of the inducible strain WT+MET3p-RAS1V13 grown on glucose at 25°C agar in the presence (repressing condition) or absence (activating condition) of methionine and cysteine. White cells from glucose agar (repressing condition) were plated. C. Switching frequencies of cells of the control strain WT+vector and the RAS1V13 overexpression strain WT+MET3p-RAS1V13 grown at 25°C in glucose liquid medium for one day and then plated on glucose or GlcNAc agar at 25°C. The colony numbers assessed ranged between 250 and 400. D. Examples of colonies on glucose and GlcNAc agar under activating conditions. E. Examples of cells from colonies on glucose and GlcNAc agar at 25°C under activating conditions. The scale bars in panel E represents 10 µm. Switching frequencies were measured after 5 days of growth at 25°C on agar.

Figure 3. The genes CDC35, PDE2, TPK1 and TPK2 play roles in GlcNAc induction of switching.

A. Switching frequencies (Switch. Freq.) of cells of the mutants cdc35/cdc35 and pde2/pde2 grown at 25°C in glucose liquid medium for 7 days (cdc35/cdc35) or 5 days (pde2/pde2), and then plated on glucose or GlcNAc agar at 25°C. The number of assessed colonies ranged between 200 and 450. B. Streaks of cells grown for 5 days of the parental control strain (WT), the mutant control strain pde2/pde2+vector and the inducible strain pde2/pde2+MET3p-PDE2 under inducing (-Met, -Cyc) and inducing (+Met, +Cys) condition. C. Switching frequencies of cells of the mutant control pde2/pde2+vector and the rescued strain pde2/pde2+MET3p-PDE2 grown in liquid glucose medium for 2 days and then plated on glucose and GlcNAc agar under activating conditions. The number of assessed colonies ranged between 350 and 450. D. Examples of colonies of pde2/pde2+vector and pde2/pde2+MET3p-PDE2 from panel C under activating conditions. E. Examples of cells from colonies in Figure D. F. Switching frequencies of parental control strains, the mutants tpk1/tpk1 and tpk2/tpk2 and the overexpression strains WT+ACTp-TPK1 and WT+ACTp-TPK2 grown in glucose medium for 5 days and then plated on glucose or GlcNAc agar. The number of assessed colonies ranged between 250 and 800 colonies.

Figure 4. WOR1, the master switch locus, is essential for GlcNAc induction and involves phosphorylation.

A. Switching frequency of the parental control and mutant wor1/wor1 at 25°C. The number assessed colonies ranged between 250 and 450. B. Examples of colonies of the control and deletion mutant wor1/wor1 grown at 25°C on GlcNAc agar. C. Switching frequencies of cells of the control WOR1/WOR1-vector and the overexpression derivatives ras1/ras1+METp-WOR1, cdc35/cdc35+METp-WOR1, pde2/pde2+METp-WOR1, tpk1/tpk1+METp-WOR1 and tpk2/tpk2+METp-WOR1 grown at 25°C in liquid glucose medium for 1 day and plated on either glucose or GlcNAc agar at 25°C under activating conditions (-Met, -Cys). The number assessed colonies ranged between 100 and 400. D. Switching frequencies of cells of the control wor1/wor1-vector, wor1/wor1+TETp-WOR1 and wor1/wor1+TETp-WOR1TA grown in glucose liquid medium at 25°C and plated on glucose or GlcNAc agar containing 50 or 200 µg per ml of doxycycline, the TET inducer, at 25°C. The number of assessed colonies ranged between 100 and 300. E. Examples of cells from colonies of cells of wor1/wor1+TETp-WOR1 and wor1/wor1+TETp-WOR1TA grown in glucose liquid medium at 25°C and plated on GlcNAc agar containing 200 µg of doxycycline at 25°C.

Figure 5. Wor1 localizes to the nucleus in the phosphorylated and unphosphorylated state.

A. GFP fluorescence of Wor1 and Wor1TA, in parallel with DAPI staining of nuclei, of strains wor1/wor1+TETp-WOR1 and wor1/wor1+TETp-WOR1TA, respectively. Cells were grown in glucose liquid medium containing 200 µg per ml of doxycycline at 25°C. B. Western blot analysis of Wor1 and Wor1TA using anti-GFP antibody in strains wor1/wor1+TETp-WOR1 and wor1/wor1+TETp-WOR1TA. Protein extracts were derived from cells grown at 25°C in glucose liquid medium containing 200 µg per ml of doxycycline.

Figure 6. A model of the regulatory circuitry involved in the induction of the white to opaque switch.

The number of plus signs and the thickness of initial pathway arrows reflect the degree of induction. Note that we have all pathways converging on the master switch gene WOR1 because of their dependency on that gene.