DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway

Abstract

Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO₂ bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

Keywords: Candida albicans; DYRK; Ras1/PKA; Yak1 kinase; dermatitis; fungal pathogen; morphogenesis.

Fig 1

Yak1 is a core regulator of hyphal morphogenesis in C. albicans. (a) Yak1 is required for C. albicans morphogenesis in response to diverse inducing cues, including growth at igh temperature (42°C, 4 hours), in 10% serum (37°C, 4 hours), in N-acetyl-glucosamine (GlcNAc; 5 mM, 37°C, 4 hours), in RPMI (37°C, 4 hours), and in Spider medium (37°C, 4 hours). Light microscopy images are representative of two biological replicates. (b) Yak1 is localized to the nucleus under both basal and filament-inducing conditions. Cells were grown for 3 hours at either 30°C or 39°C, as indicated. Cells were collected, washed twice with PBS, and fixed with 4% paraformaldehyde. Cells were then stained with 5 µg/mL of the nuclear dye, Hoescht 33342, and imaged using light and fluorescence microscopy. Data are representative of two biological replicates. (c) Y588 is required for Yak1 function in C. albicans. In response to the inducing cue of high temperature (42°C), complementation of a yak1 homozygous-deletion mutant with one allele of YAK1 rescues morphogenesis, but complementation with one allele of the phospho-ablated YAK1^Y588F does not rescue the defect. Cells were grown in YPD at 42°C for 4 hours to induce morphogenesis. Light microscopy images are representative of two biological replicates. (d) Mutation of YAK1 does not alter its expression. Cells were grown at 30°C for 3 hours. Transcript levels relative to the wild-type control were measured by RT-qPCR and normalized to ACT1. Data are presented as mean of technical quadruplicates. Measure of center represents the mean of the data, and error bars represent the standard error of the mean. Results are representative of two biological replicates. (e) Cellular level of Yak1^Y588F is comparable to that of wild-type Yak1 protein. Cells were grown at 30°C for 4 hours and then protein was extracted under denaturing conditions. Samples were run on an 8% SDS-PAGE gel and probed with an anti-FLAG antibody to assess relative protein levels. Data are representative of two biological replicates.

Fig 2

Yak1 acts downstream or parallel to PKA to induce C. albicans filamentation. (a) Homozygous deletion of genes encoding either of the PKA subunits, TPK1 or TPK2, results in a defect in C. albicans morphogenesis. Overexpression of YAK1 driven by a tetO promoter rescues the filamentation defect in both deletion-strain backgrounds. Cells were grown in YPD at 42°C for 4 hours. Light microscopy images are representative of two biological replicates. (b) Overexpression of YAK1 rescues the defect in filamentation observed with the inhibition of PKA activity. One PKA subunit gene, TPK1, was deleted and the remaining subunit gene, TPK2, was replaced with an allele encoding an analog-sensitive (AS) version of the kinase; treatment of this strain with the ATP analog 1-NM-PP1 selectively inhibits the kinase activity of TPK2. Cells were grown in YPD at 42°C in the absence or presence of 2.5 µM 1-NM-PP1 for 4 hours. Light microscopy images are representative of two biological replicates. (c) Overexpression of TPK2 driven by a tetO promoter results in a hyper-filamentous phenotype under basal conditions. Cells were grown at 34°C for 6 hours. Homozygous deletion of YAK1 in the tetO-TPK2/TPK2 background restores wild-type morphology. Light microscopy images are representative of two biological replicates. (d) Yak1 does not affect PKA regulation of glycogen storage. All strains were grown on YPD agar either with or without 2.5 µM 1-NM-PP1 for 2 days. The plate was then inverted over iodine crystals for 5 minutes to stain for glycogen content. Overexpression of YAK1 does not rescue the increase in glycogen accumulation observed in a strain of C. albicans, where TPK1 is deleted and TPK2 encodes an analog-sensitive version of the kinase that can be inhibited by the ATP analog, 1-NM-PP1. Images are representative of two biological replicates.

Fig 3

Mutation of predicted PKA phosphorylation sites in Yak1 does not inhibit its function. (a) Schematic of the five predicted PKA phosphorylation residues in C. albicans Yak1. (b) Phospho-ablation of predicted PKA phosphorylation sites S139, S212, S261, S355, or T584 does not affect the ability of C. albicans to undergo morphogenesis. Cells were grown at 39°C for 4 hours. Light microscopy is representative of two biological replicates. (c) Phospho-ablation of any of the five predicted PKA-phosphorylated residues in Yak1 does not alter protein level. Cells were grown at 30°C for 3 hours and then protein was extracted under denaturing conditions. Samples were fractionated on an 8% SDS-PAGE gel and probed with an anti-FLAG antibody. Western blot is representative of two biological replicates. (d) Phospho-ablation (5×Ablation-HF) of all five predicted PKA phosphorylation sites in Yak1 results in a hyper-filamentous phenotype. However, substitution with a phospho-mimetic residue at each of the five predicted sites in Yak1 (5×Mimetic-HF) does not alter C. albicans morphogenesis. Cells were grown at 30°C for 4 hours. Light microscopy is representative of two biological replicates. (e) Phospho-ablative or phospho-mimetic substitutions at all five predicted PKA phosphorylation sites in Yak1 do not alter protein level. Cells were grown at 30°C for 3 hours and then protein was extracted under denaturing conditions. Samples were fractionated on an 8% SDS-PAGE gel and probed with an anti-FLAG antibody. Western blot is representative of two biological replicates.

Fig 4

Yak1 is dependent on canonical transcription factors known to regulate C. albicans morphogenesis. (a) Deletion of FLO8 or EFG1 blocks filamentation, and overexpression of YAK1 does not rescue this defect in either strain background. Strains were grown in RPMI with 10% serum at 37°C for 6 hours and visualized by microscopy. Images are representative of two biological replicates. (b) Migration of Efg1 during SDS-PAGE is unaffected by the deletion of YAK1. Strains were grown at either 30°C or 39°C for 3 hours, as indicated. Protein was extracted under denaturing conditions and an anti-FLAG antibody was used to assess the relative protein level and migration position via western blot analysis. Position of a non-specific immunoreactive band is indicated by an arrow. Data are representative of two biological replicates. (c) Deletion of YAK1 results in reduced mobility of Flo8. Strains were grown at either 30°C or 39°C for 3 hours, as indicated. Protein was extracted under denaturing conditions, and an anti-FLAG antibody was used to assess migration position via western blotting. Position of a non-specific immunoreactive band is indicated by an arrow. Data are representative of two biological replicates. (d) At 30°C, overexpression of the transcription factors UME6, BRG1, or TEC1 driven by a tetON promoter system induced by doxycycline (DOX, 10 µg/mL) drives filamentation in C. albicans. Treatment with the Yak1 inhibitor 1-acetyl-beta-carboline (1-ABC; 200 µM) does not block the mutant phenotype in any of the overexpression strains. Strains were grown in the absence or presence of DOX or 1-ABC for 8 hours at 30°C. To confirm that 1-ABC could inhibit filamentation under the conditions tested, a wild-type strain was grown in YPD at 42°C for 4 hours in the absence or presence of 200 µM 1-ABC. Light microscopy images are representative of two biological replicates. (e) Homozygous deletion of the histone H3 variant gene HHT1 rescues the filamentation defect observed in a yak1 homozygous-deletion mutant. Cells were grown at 42°C for 4 hours. Light microscopy images are representative of two biological replicates.

Fig 5

Yak1 regulates C. albicans morphogenesis in an environmentally-contingent manner. (a) Yak1 is not required for filamentation under 5% CO₂. Wildtype or a yak1 homozygous-deletion mutant were grown for 6 hours in RPMI buffered with either MOPS or NaHCO₃ under either ambient or 5% CO_2, respectively. Data are representative of two biological replicates. (b) YAK1 was overexpressed in the TPK2 analog-sensitive strain (TPK2-AS/TPK2-AS tpk1Δ/tpk1Δ). In the presence of 1-NM-PP1 (2.5 µM), where PKA is inhibited, overexpression of YAK1 rescued the defect in filamentation. Strains were grown at 37°C for 3 hours in RPMI buffered with NaHCO₃ under 5% CO₂. Data are representative of two biological replicates. (c) Under ambient (room air) CO₂ concentration, overexpression of CYR1 partially rescues the defect in filamentation of a yak1-deletion strain. Addition of exogenous dbcAMP, which directly stimulates PKA, further rescues this defect to wild-type levels of filamentation. Strains were grown at 37°C for 3 hours in RPMI buffered with MOPS under ambient CO₂. Data are representative of two biological replicates. (d) Treatment with the carbonic anhydrase inhibitor, acetazolamide, restores the requirement for Yak1 for cells to filament under 5% CO₂. Strains were grown at 37°C for 3 hours in RPMI buffered with either MOPS under ambient CO₂ or NaHCO₃ under 5% CO₂, as indicated. Data are representative of two biological replicates. (e) Homozygous deletion of the predicted DYRK-encoding gene POM1 in combination with homozygous deletion of YAK1 blocks filamentation under both ambient and elevated CO₂. Cells were grown in either YPD (42°C) or RPMI buffered with either MOPS under ambient CO_2, or NaHCO₃ under 5% CO_2, at 37°C for 4 hours, as indicated. Light microscopy images are representative of two biological replicates.

Fig 6

Model for Yak1 regulation of C. albicans morphogenesis in an environmentally-contingent manner. Under ambient CO₂, Yak1 is required for C. albicans morphogenesis, where it acts downstream or parallel to PKA and it is dependent on core filamentation-regulatory transcription factors and histone H3 variant, Hht1. Under physiological concentrations of CO₂, Yak1 acts downstream or parallel to PKA but is dispensable for C. albicans hyphal morphogenesis due to hyperactivation of the Ras1/cAMP/PKA pathway. A distinct DYRK, Pom1, exhibits a synthetic interaction with Yak1, such that deletion of both kinases results in a block in C. albicans morphogenesis under elevated concentrations of CO₂. Figure made using Biorender.com.

Fig 7

Yak1 is required for invasive filamentation in a mouse model of C. albicans dermatitis. (a) The dorsal skin of neutropenic BALB/c mice was infected with either wild-type (SN95), yak1Δ/yak1Δ + YAK1, or yak1Δ/yak1Δ strains by topical application of 1 × 10⁷ cells suspended in PBS. At 72-hours post-infection, mice were euthanized, and full thickness punch biopsies of infected skin were collected. Formalin-fixed, paraffin-embedded tissue sections were stained with anti-C. albicans antibody to assess morphology and extent of tissue invasion (brown signal). Black arrows indicate hyphal C. albicans cells and red arrows indicate C. albicans persisting as yeast cells. Inset images provide a view of the indicated tissue regions at higher magnification to visualize C. albicans morphology. Images are representative of two tissue sections prepared for each of the three animals infected in the study. (b) 1-carboxylic acid beta-carboline (1-CABC) is more potent than 1-ABC in blocking filamentation. Wild-type C. albicans was grown in YPD at 42°C in the absence or presence of 1-ABC or 1-CABC for 6 hours to induce filamentation. Light microscopy images are representative of three biological replicates. (c) Neutropenic BALB/c mice were infected by topical application of either yak1Δ/Δ or yak1Δ/Δ + YAK1 C. albicans (1 × 10⁷ cells/mL) prepared in PBS with or without 42.4 µg/mL solution of 1-CABC. Topical treatment with 1-CABC or PBS was repeated daily until 72-hours post-infection when mice were euthanized, and punch biopsies were collected. Formalin-fixed, paraffin-embedded tissue sections were stained with an anti-C. albicans antibody to assess morphology and extent of tissue invasion (brown signal). Black arrows indicate hyphal C. albicans and red arrows indicate C. albicans persisting in yeast morphology. Inset images provide a view of the indicated tissue regions at higher magnification to visualize C. albicans morphology. Images are representative of two tissue sections prepared for each of the five animals infected in the study.