Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of <i>Candida albicans</i>

<div><p>The regulatory networks governing morphogenesis of a pleomorphic fungus, <i>Candida albicans</i> are extremely complex and remain to be completely elucidated. This study investigated the function of <i>C</i>. <i>albicans</i> yeast casein kinase 2 (CaYck2p). The <i>yck2Δ/yck2Δ</i> strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the <i>yck2Δ/yck2Δ</i> strain which showed significant upregulation of <i>UME6</i>, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes <i>ALS3</i>, <i>HWP1</i>, and <i>SUN41</i>, all of which are associated with morphogenesis and biofilm architecture. The <i>yck2Δ/yck2Δ</i> strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, <i>CHS2</i>, <i>CHS3</i>, and <i>CHS8</i>. Absence of CaYck2p also affected fungal-host interaction; the <i>yck2Δ/yck2Δ</i> strain had significantly reduced ability to damage host cells. However, the <i>yck2</i>Δ/<i>yck2</i>Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in <i>C</i>. <i>albicans</i>, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.</p></div

Quantitative gene expression analysis of cell wall synthase genes.

<p><b>A,</b> the relative mRNA expression of chitin synthase genes compared to wild type in the indicated strains grown in YPD at 30°C to mid log phase (** p<0.001 and *** p<0.0001). <b>B,</b> the relative mRNA expression of glucan synthase genes compared to wild type in the indicated strains grown in YPD at 30°C to mid log phase.</p

Morphology of the <i>yck2</i>Δ/<i>yck2</i>Δ strain.

<p><b>A,</b> relative mRNA expression of <i>YCK2</i> (a), <i>YCK3</i> (b), and <i>HRR25</i> (c) for the designated strains. Shown are mean ± S.D. of three biological replicates (* p<0.05, ** p<0.001). <b>B,</b> colony morphologies of the wild-type (a), the <i>yck2/yck2</i> insertion mutant (b), the <i>yck2</i>Δ/<i>yck2</i>Δ (c), and the <i>yck2</i>Δ/<i>yck2</i>Δ+<i>YCK2</i> complemented (d) strains grown on YPD plate at 30°C for 2 days. <b>C,</b> microscopic morphologies of the wild-type (a and e), the <i>yck2/yck2</i> insertion mutant (b and f), the <i>yck2</i>Δ/<i>yck2</i>Δ (c and g), and the complemented (d and h) strains grown in YPD medium for two h at 30°C and in RPMI 1640 medium at 37°C, respectively.</p

Minimum inhibitory concentrations of fluconazole and caspofungin for the <i>yck2</i>Δ<i>/yck2</i>Δ strain.

<p>Minimum inhibitory concentrations of fluconazole and caspofungin for the <i>yck2</i>Δ<i>/yck2</i>Δ strain.</p

Functional analysis of <i>YCK2</i> deletion.

<p><b>A</b> and <b>B,</b> The extent of biofilm formation by the indicated strains after 48 h in YPD, RPMI1640, YPGly, and Spider media at 30°C (A) and at 37°C (B). (* p<0.001). <b>C,</b> The extent of cell damage of endothelial cells (HUVECs), oral epithelial cells (OKF6/TERT), and vaginal epithelial cells (VK2E6/E7) after 180 min co-incubation with the indicated strains. (*, p<0.001 compared to both the wild-type strain and complemented strain).</p

Effect of <i>YCK2</i> deletion on cell wall integrity.

<p><b>A,</b> susceptibility of the indicated strains to various cell wall stressors, 2 mg/ml protamine sulfate, 0.1% SDS, 300 μg/ml congo red, and 10 μg/ml calcofluor white (CFW) after 48 h at 30°C. <b>B,</b> susceptibility of the designated strains to calcineurin inhibitors, 10 μg/ml cyclosporine A and 5 μg/ml FK506 and 5 μg/ml pyrvinium pamoate. Shown are representative images for three independent experiments with the same outcome. <b>C,</b> microscopic analysis of chitin deposition in the cell wall of the wild-type (a, e), the insertion mutant (b, f), the <i>yck2</i>Δ/<i>yck2</i>Δ (c, g), and the complemented (d, h) strains grown in YPD at 30°C and stained with 0.05% calcofluor white.</p