Figure 1.
The genome of Paracoccidioides encodes an α-pheromone.
(A) Mating pheromone signaling pathway described in S. cerevisiae [21]. (B) S. cerevisiae mating pheromone signaling pathway components and the homologous genes annotated in Paracoccidioides and A. fumigatus databases. Genes not annotated in databases are denoted N.A. (C) Alignment of α-pheromone precursor peptide sequences from Paracoccidioides strains and H. capsulatum. The Kex2 peptidase recognition site (KR) and the mature pheromone are indicated by blue and red boxes, respectively.
Table 1.
Strains used in this study.
Table 2.
Plasmids used in this study.
Figure 2.
Paracoccidioides expresses the α-pheromone and the a- and α-receptor genes independently of the mating type.
Mating gene expression of Paracoccidioides MAT1-1 strains (Pb01 and T8B1) and MAT1-2 strains (ATCC60855 and Pb03) in yeast (Y) and mycelial (M) form. Expression levels of MAT1-1 (A) and MAT1-2 (B), PBα (B, C), PREB (E, F) and PREA (G, H) genes were measured. Error bars are indicated, and asterisks show significant differences (*p<0.05 or **p<0.01).
Figure 3.
α-pheromone does not induce mating gene expression in Paracoccidioides.
Mating gene expression at different time points in P. brasiliensis ATCC60855 (A) and P. lutzii Pb01 (B) in mycelium form, upon induction with 50 µg/mL of synthetic Pbα pheromone. Basal expression levels of MAPK signaling pathway components in the mycelium form of P. brasiliensis Pb03 and ATCC60855 (MAT1-2) and P. lutzii Pb01 (MAT1-1) (C-K). Error bars are indicated, and asterisks show significant differences (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
α-pheromone activates PreB receptor and induced shmoo formation in the S. cerevisiae model.
Shmoo formation in S. cerevisiae BY4741 induced with 20 µg/ml synthetic MFα pheromone after 0 and 5 hrs induction (A and B), and S. cerevisiae AGLPreB induced with 40 µg/ml synthetic Pbα pheromone after 0 and 5 hrs induction (C and D). Quantification of shmoo formation using S. cerevisiae BY4741 stimulated with MFα (2 µg/mL) or S. cerevisiae AGLPreB stimulated with Pbα (4 µg/mL) (E). Induction of S. cerevisiae AGLPreB using Pbα pheromone extracted from P. lutzii Pb01 monoculture or Paracoccidioides co-cultures (Pb01/Pb03 and Pb01/Pb60855), Pbα pheromone extracted from AGMPbα culture, as well stimulation with the synthetic Pbα pheromone (40 µg/mL) (F). Error bars are indicated, and asterisks show significant differences (*p<0.05, ***p<0.001).
Figure 5.
α-pheromone activates PreB receptor, inducing growth and cell cycle arrest in the S. cerevisiae model.
Halo formation in S. cerevisiae AGLPreB stimulated with Pbα pheromone extracted from P. lutzii Pb01 monoculture (A); stimulated with 0.8, 4.0 and 40 µg synthetic Pbα pheromone (B–D respectively) or Pbα pheromone extracted from S. cerevisiae AGMPbα (E). S. cerevisiae BY4741 stimulated with 10 µg synthetic MFα (F) and with 20 µg synthetic Pbα (G). Quantification of growth inhibition zones (cm2) upon exposure to α-pheromones. Halos were recorded after 24 hrs of incubation and standard deviations indicated by error bars (H). Cell cycle arrest analysis for S. cerevisiae BY4741 upon addition of synthetic MFα (20 µg/ml) (I) and S. cerevisiae AGLPreB upon addition of synthetic Pbα pheromone (40 µg/ml) (J). (K–N) FCM profiles of α-pheromone-induced cell cycle arrest in haploid cells showing n and 2 n nuclear DNA content: BY4741 induced with 20 µg synthetic MFα (K–L) and S. cerevisiae AGLPreB induced with 40 µg synthetic Pbα pheromone (M–N). Growth rate evaluation of either S. cerevisiae BY4741 (O) or AGLPreB (P) upon addition of the cognate synthetic α-pheromone. Error bars are indicated, and asterisks show significant differences (*p<0.05, **p<0.01).
Figure 6.
α-pheromone and its cognate receptor functionally complement mating in the corresponding S. cerevisiae mutants.
Mating efficiency of S. cerevisiae null mutants, expressing Paracoccidioides α–pheromone (AGLPbα and AGMPbα) and its cognate receptor (AGLPreB), crossed at different ratios after incubation for 5 hrs (A). (B) Mating efficiency after 5 hrs incubation of S. cerevisiae strains AGΔScα and AGLPreB, upon the addition of different concentrations of Paracoccidioides synthetic α-pheromone. Error bars are indicated, and asterisks show significant differences (*p<0.05).