Epigenetic control of pheromone MAPK signaling determines sexual fecundity in Candida albicans

Abstract

Several pathogenic Candida species are capable of heritable and reversible switching between two epigenetic states, "white" and "opaque." In Candida albicans, white cells are essentially sterile, whereas opaque cells are mating-proficient. Here, we interrogate the mechanism by which the white-opaque switch regulates sexual fecundity and identify four genes in the pheromone MAPK pathway that are expressed at significantly higher levels in opaque cells than in white cells. These genes encode the β subunit of the G-protein complex (STE4), the pheromone MAPK scaffold (CST5), and the two terminal MAP kinases (CEK1/CEK2). To define the contribution of each factor to mating, C. albicans white cells were reverse-engineered to express elevated, opaque-like levels of these factors, either singly or in combination. We show that white cells co-overexpressing STE4, CST5, and CEK2 undergo mating four orders of magnitude more efficiently than control white cells and at a frequency approaching that of opaque cells. Moreover, engineered white cells recapitulate the transcriptional and morphological responses of opaque cells to pheromone. These results therefore reveal multiple bottlenecks in pheromone MAPK signaling in white cells and that alleviation of these bottlenecks enables efficient mating by these "sterile" cell types. Taken together, our findings establish that differential expression of several MAPK factors underlies the epigenetic control of mating in C. albicans We also discuss how fitness advantages could have driven the evolution of a toggle switch to regulate sexual reproduction in pathogenic Candida species.

Keywords: mating; phenotypic switching; sexual reproduction; signaling bottlenecks; transcriptional regulation.

Fig. 1.

Comparison of the pheromone MAPK pathway in C. albicans white and opaque cells. (A) Schematic of the C. albicans pheromone MAPK pathway in MTLa cells. Components highlighted in yellow are those that are transcriptionally up-regulated in opaque cells relative to white cells. (B) Analysis of RNA-sequencing data (36) indicates that four pheromone MAPK components—STE4, CST5, CEK1, and CEK2—are expressed at more than threefold higher levels in C. albicans opaque cells relative to white cells.

Fig. 2.

Quantification of mating in C. albicans white cells ectopically expressing pheromone MAPK components. (A) Representative images of mating projection formation following coincubation of a and α cells that were white-locked (white control) and white-locked and overexpressing CEK2/CST5/STE4 (white + CEK2/CST5/STE4) or wild-type opaque (opaque control). Cells were coincubated on Spider medium for 24 h and imaged. Asterisks indicate cells with mating projections. (Scale bar, 5 μm.) (B) Quantification of mating projections in mixtures of white-locked a and α cells overexpressing MAPK genes, either singly or in combination, compared with white-locked parental cells (white control) or opaque cells (opaque control). Significant differences are relative to the white control. ^#P < 0.05, Kruskal–Wallis (Mann–Whitney pairwise). n = 3 biological replicates. Error bars = SD. (C) Conjugation frequencies between white-locked a and α cells overexpressing MAPK genes, either singly or in combination, compared with white-locked cells (white control) or opaque cells (opaque control). Significant differences are relative to the white-locked control. ^###P < 0.001, ^####P < 0.0001, Kruskal–Wallis (Mann–Whitney pairwise). n = minimum of six biological replicates. Error bars = SEM. (D) Scanning electron micrographs of white-locked a cells (white control), white-locked a cells overexpressing CEK2, CST5, and STE4 (white + CEK2/CST5/STE4), and opaque a cells. Cells were grown under nonmating, vegetative conditions. (Scale bar, 2 µm.)

Fig. 3.

Comparison of expression profiles between mating mixes of white cells (Wh), white cells engineered to overexpress MAPK genes (Wh-OE), and opaque cells (Op). Expression levels of genes involved in (A) the core pheromone MAPK-signaling cascade; (B) pheromone production and processing; (C) cell polarization, cell fusion, and karyogamy; and (D) opaque-enriched genes. In all cases, mating mixes of a and α cells were incubated on Spider medium for 8 h, and RNA expression was determined. Data are normalized to expression levels in mating mixes of control white cells. White cells are white-locked cells (wor1Δ/wor1Δ), and engineered white-locked cells are constitutively expressing STE4, CST5, and CEK2 (white-OE cells). Opaque-enriched genes represent those most highly up-regulated in opaque cells relative to white cells (36). Average of two biological replicates.

Fig. 4.

Comparison of protein levels for pheromone MAPK components in white cells, engineered white cells, and opaque cells. Western blots were performed for strains expressing epitope-tagged versions of Cek2 (A), Cst5 (B), or Ste4 (C). Op: mating crosses between wild-type opaque a and α cells; Wh: mating crosses between wor1Δ/wor1Δ a and α cells. White-OE: mating crosses between white-locked a and α cells that ectopically express CEK2 (A), CST5 (B), or STE4 (C). MAPK protein levels are relative to control white cells (normalized to tubulin signal). Cek2 and Ste4: n = 4 biological replicates. Cst5: n = 2 biological replicates. Graphs show mean ± SEM. See SI Appendix, Fig. S5, for Western blot images.