The Cbk1-Ace2 axis guides Candida albicans from yeast to hyphae and back again

Abstract

Since its description in S. cerevisiae, the Regulation of Ace2 and Morphogenesis (RAM) pathway has been studied for nearly 20 years in multiple model and pathogenic fungi. In pathogenic fungi, the RAM pathway carries out many functions through mechanisms that remain to be defined in detail. Recently, we reported that Cbk1-mediated phosphorylation of the transcription factor Ace2 functions to repress the hyphae-to-yeast transition in Candida albicans. This transition is understudied relative to the yeast-to-hyphae transition. Subapical hyphal cell compartments are arrested in G1 until the point at which lateral yeast emerge. Here, we discuss this model and report new data indicating that a second G1 associated protein, the mitotic exit regulator Amn1. In S. cerevisiae diploid cells, Amn1 negatively regulates Ace2 at both the gene expression level through a negative feedback loop and at the protein level by targeting Ace2 for degradation. In C. albicans, Amn1 and Ace2 also form a feedback loop at the level of gene expression. Deletion of AMN1 decreases lateral yeast formation relative to wild type in maturing hyphae and is associated with decreased expression of PES1, a positive regulator of lateral yeast formation. These data indicate that the regulation of mitotic exit plays a role in determining the timing of lateral yeast emergence from hyphae in C. albicans. We also propose an integrated model for the interplay between the Cbk1-Ace2 axis and other hyphal stage regulators during the process of filamentation and transition back to yeast.

Keywords: Candida albicans; G1; Hyphae; Lateral yeast; Mitotic exit; RAM network.

Fig. 1

Candida albicans forms multiple morphological cells types. a Examples of yeast, pseudohyphal, and hyphal C. albicans cells. b Lateral yeast formation in mature hyphal cells. c Schematic indicating cell cycle state of sub-apical cells and the transition to lateral yeast formation

Fig. 2

Regulation of Ace2 and Morphogenesis (RAM) pathway and Cbk1 phosphosites of Ace2. a Schematic showing the different components of the RAM pathway and examples of three Cbk1-regulated transcription factors in C. albicans. b The location of Cbk1 consensus phosphorylation sites and the Cbk1 dependent functions that were identified by mutation of those sites to non-phosphorylatable alanine (Wakade et al. 2020)

Fig. 3

Transcriptional interplay between Ace2 and the G1 regulator Amn1 in C. albicans. a AMN1 expression is decreased in ace2∆∆ in both yeast and hyphal phase as determined by RNA-sequencing experiments (Wakade et al. 2020). The effect was confirmed for hyphae using RT-PCR. b Deletion of AMN1 increases the expression of Ace2 targets CHT3 and SCW11 in hyphae. c Lateral yeast emergence is delayed in amn1∆∆ mutants relative to wild type following hyphal induction in Spider medium at 37 °C. d Micrographs of WT and amn1∆∆ strains induced in Spider medium as for (c) showing that the deletion mutant forms true hyphae. e The expression of PES1 was determined by RT-PCR 4 and 8 h after hyphal induction with Spider medium at 37 °C. For a, b and e bars indicate the mean ratio of the expression of the indicated gene expressed as fold-change in the ratio of mutant/wild type. Error bars indicate the standard deviation of two biological replicates performed in duplicate. Significant differences were determined by Student’s t test with a threshold of P < 0.05: *P < 0.05; **P < 0.01; ***P < 0.001. Bars indicate the number of hyphae with lateral yeast as determined by light microscopy. For c, at least 100 cells were characterized per replicate. The error bars indicate the standard deviation for at least two independent experiments. Student’s t test analysis of the difference between the wild type and amn1∆∆ cells indicated it was statistically significant (P < 0.05)

Fig. 4

Amn1 and Ace2 form a feedback loop at the transcriptional level and regulate PES1 expression. a ACE2 expression is increased in amn1∆∆ mutants during hyphae induction. Bars indicate the mean ratio of the expression of the indicated gene expressed as fold-change in the ratio of mutant/wild type. Error bars indicate the standard deviation of two biological replicates performed in duplicate. Significant differences were determined by Student’s t test with a threshold of P < 0.05: **P < 0.01. b Data from Fig. 3a/4a and previously published data showing that Ace2 represses PES1 expression during hyphae formation (Wakade et al. 2020) provides genetic support for the indicated feedback loop

Fig. 5

Schematic for the Cbk1-Ace2 axis during hyphal morphogenesis. Green arrows indicate activating interactions and red symbols indicate repressing interactions. The presence of a P indicates phosphorylation of the indicated protein by Cbk1