The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Abstract

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Fig 1. 3-MB-PP1 inhibits the activity of analog-sensitive Ssn3^AS, but not Ssn3^WTin vitro.

In vitro kinase reactions contained purified Mediator from a strain with Ssn3^WT or Ssn3^AS, ³²P-ATP, purified GST-tagged RNA Pol II C-terminal domain (CTD) and the indicated concentrations of 3-MB-PP1 inhibitor. Reactions were analyzed by SDS-PAGE and visualized by phosphorimaging.

Fig 2. 3-MB-PP1 stimulates hyphal growth in a strain bearing analog-sensitive alleles of SSN3.

A. Morphology of SC5314 (WT), ssn3^AS and ssn3^KD strains was assessed after growth in YNBAG supplemented with either 5 μM 3-MB-PP1 or vehicle (DMSO) for 3h at 30°C. Quantification of yeast, pseudohyphae and hyphae in cultures was performed by microscopic analysis of blinded samples. Results from ANOVA with multiple comparisons for the percent of cells as hyphae is shown; ****, p<0.001, ns, not significant. Lower case letters indicate statistically significant differences in comparison to a) WT with 3MB-PP1 at p<0.001 or b) to WT with DMSO at p<0.001. B. Representative images of cell populations from cultures analyzed in panel A are shown.

Fig 3. Heat map for genes induced upon inhibition of Ssn3^AS by 3-MB-PP1.

Transcripts for seventy-three genes were 2-fold higher (p<0.05, FDR corrected) in both the comparison of Ssn3^AS with 3-MB-PP1 compared to WT with 3-MB-PP1 and of Ssn3^AS with 3-MB-PP1 compared to with the DMSO control. Genes are listed in order with the greatest magnitude difference between Ssn3^AS with and without 3-MB-PP1 at the top. The heat map shows three replicates per sample of Log2-transformed counts per million for expressed transcripts.

Fig 4. Overview of proteins for which two phosphopeptides were lower by >2 fold upon inhibition of Ssn3.

Focusing on peptides that were significantly lower in Ssn3^AS treated with 3-MB-PP1 relative to control cultures (p<0.05 FDR-corrected), we found 977 peptides that were 2-fold lower upon 3-MB-PP1 inhibition of ssn3^AS relative to changes in the wild type. Two hundred and eighteen proteins had two more peptides that met these criteria, forty one of which were predicted to have nuclear localization. Med4 is a validated C. albicans Ssn3 target. If an alias is available for an unnamed gene, it is shown in parentheses.

Fig 5. Ssn3 repression of filamentation is FLO8 dependent.

A. Colony morphology of SC5314 wild type (WT), ssn3Δ/Δ, flo8Δ/Δ, and ssn3Δ/Δflo8Δ/Δ strains grown on YNBA agar medium alone or supplemented with 110 mM glucose at 30°C or on YNBA at 37°C. B. Cell morphology of strains tested in (A) after growth in YNBN_2.5AG (11 mM glucose) at 30°C for 3 hours. C. NanoString analysis of indicated hypha-associated genes in WT, ssn3Δ/Δ, and ssn3Δ/Δflo8Δ/Δ. RNA was extracted from cells grown as indicated for (B) but for 75 minutes. Expression levels was represented by mean and standard deviation after normalization to TEF1 and ACT1 reference transcripts. Expression of each gene was normalized to levels for the WT. Significant differences from WT are indicated as **, p<0.01 and ***,p<0.001. D. Embedded colonies of the same set of strains shown in (A) grown in YPS agar at 25°C.

Fig 6. Alteration of Flo8 phosphosite residues influences Flo8 function.

A.Wild type (WT), ssn3Δ/Δ, flo8Δ/Δ and ssn3Δ/Δflo8Δ/Δ strains were imaged after growth as colonies on YNBA or YNBA+110 mM glucose at 37°C. In addition, ssn3Δ/Δflo8Δ/Δ or flo8Δ/Δ were complemented with constructs that encoded Flo8^WT, Flo8^T589A/S620A or Flo8^T589E/S620E with C-terminal 3XHA tags. B. Cell morphology of WT, flo8Δ/Δ, and flo8Δ/Δ expressing 3XHA-tagged Flo8^WT, Flo8^T589A,S620A or Flo8^T589E,S620E after growth in YNBN_2.5AG(11 mM glucose) at 30°C for 3h. C. Expression of hypha-associated transcripts in flo8Δ/Δ expressing either 3XHA-tagged Flo8^WT or Flo8^T589A/S620A after growth in YNBNAG for ~2h^. Data are normalized levels of the corresponding transcript in flo8Δ/Δ. Data show the mean and standard deviation from measurement on triplicate RNA samples. P-values, determined by t-test, indicated by *, p<0.05; **, p<0.01; ***, p<0.001; or the specific value was indicated. D. Immunoblotting analysis on 10% SDS-PAGE with an α-HA antibody shows levels and mobility of 3X-HA derivatives of Flo8, Flo8^T589A/S620A or Flo8^T589E/S620E in either WT or ssn3Δ/Δ. CBS indicates levels of Coomassie Blue stained total protein in a different region of the blot and is used as a loading control. E. Immunoblot of Flo8-HA levels and mobility on 10% SDS-PAGE in strains with full Ssn3 activity, WT, ssn3^AS, ssn3Δ/Δ (Δ/Δ) or ssn3^KD (which expresses the kinase dead variant; kd/kd). CBS indicates levels of Coomassie Blue stained total protein in the region of the target protein, and is used as a loading control.

Fig 7. Medium alkalinization by Ssn3 is dependent on Stp2, Flo8, and Efg1.

Medium pH was assessed on YNBA with increasing concentrations of glucose; bromocresol purple was added as a pH indicator. All cultures were incubated at 37°C. A. Comparison of medium pH upon growth of SC5314 (WT), ssn3Δ/Δ, stp2Δ/Δand ssn3Δ/Δstp2Δ/Δstrains. B. WT, ssn3Δ/Δ, flo8Δ/Δ and ssn3Δ/Δflo8Δ/Δ alone or complemented with C-terminally 3XHA-tagged Flo8^WT or Flo8^T589A/S620A were grown as described above. C. WT, ssn3Δ/Δ, flo8Δ/Δ and ssn3Δ/Δflo8Δ/Δ were compared to efg1Δ/Δ, ssn3Δ/Δefg1Δ/Δ, flo8Δ/Δefg1Δ/Δ, and ssn3Δ/Δflo8Δ/Δefg1Δ/Δ strains.

Fig 8. Summary of SSN3-centered genetic interactions revealed by this study.

Hyperfilamentation of ssn3Δ/Δ depends on Flo8 under all conditions tested including in liquid, on agar, and in embedded conditions (orange arrow). Flo8 activity in filamentation assays is tuned at T589 and S620, which were identified as sites phosphorylated by Ssn3. Efg1 plays positive roles in the hyperfilamentation of the ssn3Δ/Δmutant (blue arrow) but plays a negative role in filamentation in embedded conditions (blue block). Deletion ofSSN3also leads to hyperalkalinization of amino acid-rich media, and alkalinization is not required for filamentation. Alkalinization is reduced or no longer observed in ssn3 mutants lacking FLO8, EFG1 or STP2. Future studies will determine the mechanisms by which Ssn3 impacts metabolism through Flo8 and Efg1, if it directly modulates Efg1 levels (grey, dotted block arrow) and if Ssn3, Flo8 or Efg1 affect Stp2-dependent metabolism (grey dotted arrow).