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. 2014 Oct 2;10(10):e1004567.
doi: 10.1371/journal.pgen.1004567. eCollection 2014 Oct.

Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation

Allia K Lindsay  1 , Diana K Morales  1 , Zhongle Liu  2 , Nora Grahl  1 , Anda Zhang  2 , Sven D Willger  1 , Lawrence C Myers  2 , Deborah A Hogan  1
Affiliations

Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation

Allia K Lindsay et al. PLoS Genet. .

Abstract

Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module-Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Primary role of Mediator modules in transcription.
The Mediator complex is composed of a Core module (broken lines) and a Cdk8 module (solid blue line). The Core module is sub-divided into a head (green), middle (pink) and tail (orange) domain; each domain is composed of multiple subunits, and is primarily involved in activation of transcription. In contrast, the Cdk8 module (blue)—composed of Ssn3, Ssn8, Srb8 and Srb9—is primarily implicated in transcriptional repression. RNA Pol II: RNA polymerase II; TFs: transcription factors.
Figure 2
Figure 2. Genetic screen revealed that ssn3 and ssn8 mutants maintain wrinkling in the presence of PYO.
Spot-inoculated colonies of wild type (SC5314), ssn3Δ/Δs, ssn3Δ/Δ+SSN3 (complemented mutant), ssn8Δ/Δ and ssn8Δ/Δ+SSN8 (complemented mutant) were grown on YNBAG10N-agar at 37°C (wrinkling-inducing conditions) in the presence of vehicle or 20 µM PYO. Strains were also grown on YNBG100-agar at 30°C (yeast growth conditions). Colonies were imaged with a dissecting stereoscope after 48 h of growth.
Figure 3
Figure 3. The Ssn3-Ssn8 cyclin kinase-cyclin pair influences sensitivity to PYO via a phosphorylation event.
Colonies of wild type (SC5314), ssn3Δ/Δ, ssn3Δ/Δ+SSN3 and the ssn3Δ/Δ+ssn3D325 kinase dead mutant were grown on YNBAG10N-agar with vehicle or 20 µM PYO at 37°C for 48 h and then imaged with a dissecting stereoscope.
Figure 4
Figure 4. The stability of Ssn3 and Ssn8 is unaffected by PYO.
Strains with both alleles of SSN3 or SSN8 encoding HA-tagged variants of the native protein were grown at 37°C in YNBG10NP and supplemented with uridine in the presence of vehicle or 20 µM PYO. Cultures were harvested for protein extraction at mid-exponential phase, when the OD600 nm was ≈0.5. Calculated molecular weights for HA-tagged Ssn3, HA-tagged Ssn8 and tubulin are 73 kD, 54.4 kD and 50 kD and weight estimates, relative to the molecular weight standards, are 79 kD, 55 kD and 54 kD respectively.
Figure 5
Figure 5. The ssn3 mutant hyperalkalinizes the extracellular milieu even in the presence of PYO.
Colonies of wild type (SC5314), ssn3Δ/Δ and ssn3Δ/Δ+SSN3 were grown on YNBAG10N-agar containing 0.01% bromocresol purple in the presence of vehicle or 20 µM PYO for 48 h at 37°C and then imaged with a digital camera.
Figure 6
Figure 6. Loss of SSN3 results in increased expression of glycolysis genes and glucose transporters.
Wrinkled colonies of wild type (WT; SC5314), ssn3Δ/Δ (Δ/Δ) and ssn3Δ/Δ+SSN3 (SSN3) were grown on YNBAG10N-agar for 24 h. Transcript levels of (A) HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, PGK1, GPM1, GPM2, ENO1 and CDC19 and (B) HGT2 and HXT5 were measured by qRT-PCR and normalized to the control transcript PMA1. Error bars represent SEM (A: n = 3; B: n = 2); * indicates genes upregulated with p≤0.05 while **denotes p≤0.01. Unless otherwise indicated, comparisons were made to the respective wild type.
Figure 7
Figure 7. Absence of Ssn3 results in increased glucose transport.
Wild type (SC5314) and ssn3Δ/Δ were cultured at 30°C for 6 h in YNBAG10P (yeast growth conditions). Supernatants were then collected for HPLC analysis of glucose consumption. All data were normalized to OD600 nm, and error bars represent SEM (n = 3); * indicates p≤0.05 compared to the wild type.
Figure 8
Figure 8. The increased oxidative metabolism of the ssn3 mutant directly correlates with wrinkled colony formation.
Wild type (WT; SC5314), ssn3Δ/Δ and ssn3Δ/Δ+SSN3 were cultured at 30°C for 6 h in YNBG100P. alamarBlue reduction was measured (A) in the absence of treatment and (B) with exposure to 0, 20 or 100 µM PYO. The reduction of alamarBlue, presented as arbitrary units (AU) was normalized to the OD600 nm culture as described in the Methods section. Error bars represent SEM (n = 3); * = p≤0.05, ** = p≤0.01, *** = p≤0.001 with respect to wild type at the respective time point (A), the vehicle-treated wild type (B) or, where indicated, the 20 µM PYO-treated wild type (B). (C) Colonies of wild type (SC5314) and ssn3Δ/Δ were grown on YNBAG10N-agar containing 0.01% bromocresol purple in the presence of 0, 20, 50 or 100 µM PYO for 48 h at 37°C and then imaged with a digital camera. Error bars represent SEM (n = 3); * = p≤0.05, ** = p≤0.01, *** = p≤0.001 with respect to wild type at the respective time point (A), the vehicle-treated wild type (B) or, where indicated, the 20 µM PYO-treated wild type (B).
Figure 9
Figure 9. The ssn3 mutant contains higher levels of ATP in the presence and absence of PYO.
ATP levels of wild type (WT; SC5314), ssn3Δ/Δ (Δ/Δ) and ssn3Δ/Δ+SSN3 (SSN3) were measured following 6 h incubation at 37°C in YNBAG10NP and treated with vehicle or 20 µM PYO. Error bars represent SEM (n = 3); * = p≤0.05, ** = p≤0.01 and *** = p≤0.001. Unless otherwise indicated, comparisons were made to the respective wild type control. ATP levels are directly proportional to the luminescent signal which is stated in relative light units (RLU) normalized to OD600.
Figure 10
Figure 10. Mutation of SSN3 increases biofilm formation.
Wild type (SC5314), ssn3Δ/Δ and ssn3Δ/Δ+SSN3 were grown at 37°C on serum-soaked silicone squares submerged in YNBG100. After 48 h had elapsed, biofilms were dried in an oven and then weighed. Error bars represent SEM (n = 2); * = p≤0.05 with respect to wild type.
Figure 11
Figure 11. Phenotypes related to defects in the Cdk8 module can be multifactorial.
The Cdk8 module of Mediator negatively regulates glucose transport, glycolysis and respiratory activity, with the latter two being exacerbated by treatment with PYO. This module positively regulates NADPH production via the PPP, which partly explains the increased sensitivity of Cdk8 mutants to oxidative stress. Ultimately, biofilm formation is associated with increased infection and adverse patient prognosis.
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