The Cst20 and Ptk2 kinases are involved in hyphal steering and thigmotropic responses in Candida albicans.

Angela Maria Lopez Garcia1, Mariana C. Almeida1, Tina Bedekovič 1 and Alexandra C. Brand1
1 University of Aberdeen, MRC Centre for Medical Mycology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK


Objectives:  Deletion of Ptk2 or mutation of Cst20 in Candida albicans result in contrasting hyphal phenotypes.  Our hypothesis is that they function antagonistically during directional hyphal growth.  Our objective is to elucidate the functions of these protein kinases in the development and directional growth of Candida albicans hyphae. 
Methods: Using the CRISPR-Cas9 gene editing system, deletion mutants, ptk2Δ and cst20Δ, were generated. The kinase domain was eliminated in Cst20 (Cst201-549), which retained the CRIB domain. Yeast cells were spotted on rich YPD agar medium supplemented with cell-stressing compounds: calcofluor white (100 µg/ml), , SDS (0.03 %), or Hygromycin B (400 µg/ml). Hyphal morphology was induced in RPMI containing L-glutamine, 20 % fetal bovine serum +/- glucose (2%) or Modified Soll’s Medium at 37 °C and β-Glucan exposure was evaluated. Deletion mutants were analysed by transmission microscopy. Microfabricated chambers with topological features were used to investigate the hyphal response to thigmotropic cues. Hyphae were induced in 20 % serum, 2 % glucose for 2 h. To visualise interactions with human cells, epithelial cells were co-incubated with C. albicans wild-type or mutant strains for 3 h at 37 ºC in 5 % CO2 and analysed using a spinning disk microscope. 
Results:  Disruption of PTK2, CST20 or the Cst20 kinase domain did not affect hypha formation under our tested conditions. Cells lacking Ptk2 were sensitive to SDS but resistant to high concentrations of Hygromycin B (400µg/ml) and Calcofluor white compared to wild-type cells. In contrast, the cst20Δ mutant was sensitive.  Deletion of PTK2 produced kinked hyphae with partial loss of contact-dependent (thigmotropic) steering responses. Truncation of the kinase domain of Cst20 resulted in straight hyphae that were unresponsive to all directional cues tested, including an inability to alter trajectory and follow contours during growth on host cells. Deletion of Ptk2 or Cst20 affected cell wall thickness
Conclusion: Our results tentatively support the view that the Cst20 and Ptk2 kinases operate antagonistically in the C. albicans hyphal steering system that influences the Cdc42-based cell polarity machinery.  We hypothesise that the N-terminal CRIB domain of Cst20Δ1-549 sequesters a small GTPase whose cycling is required for hyphae to adjust their direction of cell growth.  This interaction may result in the completely straight growth phenotype of this mutant.