White cells facilitate opposite- and same-sex mating of opaque cells in Candida albicans

Abstract

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.

Figure 1. White a cells induce mating projection formation in opaque α cells in mixed cultures of white (a) and opaque (α) cells.

Strains: wor1Δ/Δ (GH1248, MTLa/a); wor2Δ/Δ (MMY627, MTLa/Δ). (A) Cellular images of single strain cultures. WTα1, GH1349, MTLα/α; WTα2, SN152α, MTLΔ/α. Five WT a strains of different background (WTa1 to WTa5) and the wor1Δ/Δ and wor2Δ/Δ mutants were used. SZ306, MTL a/α, served as a control. (B) Cellular images of mixed cultures. 4×10⁶ opaque α cells (GH1349) were mixed with 4×10⁶ cells of different background as indicated. The mixtures were spotted onto Lee's glucose medium and cultured at 25°C in air for 24 hours. Cellular images and percentages of opaque α cells (GH1349) with mating projections are shown. Cells with at least one mating projection were counted. The mixture of opaque α cells (GH1349) and opaque a cells (GH1012) served as a positive control. The percentage of opaque α cells (GH1349) with mating projections in the opaque a×α mixture is not shown since both α and a cells formed mating projections. SZ306 (a/α) and SN152α served as negative controls. NA, not available. W (or wh), white; O (or op), opaque; P, mating projection. Scale bar, 10 µm.

Figure 2. MFA1 expression in white a cells.

Two reporter strains GH1600 (SZ306MFA1p-GFP) and GH1603 (wor1Δ/ΔMFA1p-GFP), in which GFP gene was under the control of the MFA1 promoter, were used. wh, white; op, opaque. Strains used: GH1600 and GH1603. (A) α-Pheromone induces MFA1 expression in white a cells. Cells were first grown at 25°C for 36 hours to stationary phase and inoculated into fresh Lee's glucose medium (1×10⁷ cells/ml). α-Pheromone peptide was added every two hours to the cultures over an eight-hour period. The final concentration of α-pheromone peptide was 8×10⁻⁶ M. Expression of GFP proteins was examined with a fluorescence microscope. Opaque a cells of strain SZ306MFA1p-GFP served as a control. Images of untreated cells are shown in top panels. “−α-ph”, no α-pheromone added (upper panel); “+α-ph”, α-pheromone added (lower panel). BF, bright field. Scale bar, 10 µm. (B) Opaque α cells induce MFA1 expression in white a cells. 4×10⁶ white a cells of each tester strain indicated were mixed with equal number of opaque α cells (GH1349 MTLα/α). The mixtures were spotted onto Lee's glucose medium and incubated at 25°C for 24 h. Expression of GFP proteins in white cells of the tester strains was examined with a fluorescence microscope. Images of single strain cultures (white cells of the tester strains) are shown in the upper panel. Mixed cultures are shown in the lower panel. BF, bright field. Scale bar, 10 µm.

Figure 3. a-Pheromone (MFa1) and the α-pheromone response pathway (Ste2-MAPK-Cph1) of a cells are required for a white cells to induce mating projection formation in α opaque cells (GH1349).

Strains used: GH1013, GH1609, GH1610, GH1613, GH1614, and GH1615. (A) Cellular images of single strain cultures. (B) Cellular images of mixed cultures. Opaque α cells (GH1349) were mixed with white a cells of different mutants as indicated. This experiment was performed as described in Figure 1B. Percentages of opaque α cells (GH1349) with mating projections are shown below the images. W, white; O, opaque; P, mating projection. Scale bar, 10 µm.

Figure 4. The a-pheromone response pathway (Ste3-MAPK-Cph1) of α opaque cells are required for a white cells (WTa, SN152a/Δ) to induce mating projection formation in α opaque cells.

Strains used: SN152a, GH1349, GH1611, GH1616, and GH1247. (A) Cellular images of single strain cultures. (B) Cellular images of mixed cultures. White cells of the WTa strain were mixed with opaque α cells of different mutants as indicated. The assay of mating projection formation was performed as described in Figure 1B. Percentages of opaque α cells with mating projections are shown below the images. The mixed culture of opaque α cells (GH1349) and white a cells (WTa) served as a positive control. W, white; O, opaque; P, mating projection. Scale bar, 10 µm.

Figure 5. White a cells induce mating projection formation in opaque α cells in a mouse skin infection model.

1×10⁷ opaque α cells (GH1349) and 1×10⁷ white “helper” cells were mixed and spotted onto the skin of newborn mice for infection. The wor1Δ/Δ mutant (SZ306w-a), WT “a” (P37005 and SZ306a) and “a/α” strains (SZ306) were used for induction of mating projections. Opaque α cells (GH1349) alone (no white “helper” control) was used as a control. Representative SEM images are shown. W, white cells; O, opaque cells; P, mating projections. Scale bar, 10 µm.

Figure 6. Schematic models for the role of white cells in opposite- and same-sex mating of opaque cells.

(A) The majority of white cells (a) secrete pheromone to facilitate opposite- and same-sex mating of the minority opaque cells. (i) When the majority are a/α cells, sexual mating between the minority of opaque a and α cells is rare. Opaque α cells secrete α-pheromone constitutively, while opaque a cells do not secrete a-pheromone without exposure to a certain level of α-pheromone. When both opaque a and α cells are rare, the threshold for pheromone and mating responses is typically not reached. (ii) When the majority are white a cells, sexual mating between the minority opaque a and α cells can occur. Opaque α cells secrete α-pheromone and induce the majority of white a cells to secrete a-pheromone. Thus, a-pheromone in turn induces opaque α cells to form long mating projections, which facilitate them to reach opaque a mating partners. (iii) White a/α cells do not induce mating projections and do not facilitate same-sex mating of opaque α cells. (iv) White a cells facilitate same-sex mating of opaque α cells. Opaque α cells secrete α-pheromone and induce white a cells to secrete a-pheromone. In turn, a-pheromone induces opaque α cells to form mating projections and undergo same-sex mating. (B) Schematic model depicting the crosstalk between white and opaque pheromone response pathways. First, opaque α cells constitutively secrete α-pheromone and activate the white pheromone response pathway to induce a-pheromone secretion in a cells. Then, a-pheromone secreted by white a cells activates the opaque pheromone response pathway to promote mating projection formation in opaque α cells.

Figure 7. Schematic models depicting that white cells facilitate same-sex mating of opaque cells.

(A) When the majority are white a and/or α cells, same-sex mating between the minority opaque α and α cells can occur. (1) Opaque α cells secrete α-pheromone and (2) induce the majority white a cells to secrete a-pheromone. (3) a-Pheromone in turn induces opaque α cells to form long mating projections and undergo same-sex mating. (4) a-Pheromone induces white α cells to secrete α-pheromone. (5) α-Pheromone further activates white a cells to secrete a-pheromone. (B) When the majority are a/α cells, the minority of opaque α cells cannot form mating projections and cannot undergo same-sex mating. Opaque α cells secrete α-pheromone constitutively, while a/α cells do not secrete pheromone. (C) In the absence of white a cells, opaque α cells cannot form mating projections and cannot undergo same-sex mating.