Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis
Figure 5
Msb2 shedding responds to a wide variety of environmental cues both under liquid culture and solid surface growth conditions.
A. Overnight cultures of Msb2-HA cells were grown under germination or non-germination conditions, washed, and resuspended in fresh media under different stress conditions: osmotic stress (1 M Sorbitol, 1 M NaCl), cell wall stress (100 µg/ ml CFW), and oxidative stress (5 mM H2O2) for 1 h. Msb2 shedding was determined by immunoblotting of cell supernatants. B. Msb2 shedding on solid surfaces differs between colony centers and peripheries. Msb2-HA strain was grown on nitrocellulose membrane overlaid on YNB + 1.25% NAG +1% agar containing the stress inducing agents in (A), as well as 100 µg/ ml of Congo red. After 3 days, colony morphology was documented and nitrocellulose membranes were washed, blocked, and immunoblotted with anti-HA antibody for Msb2 shedding determination (top). The intensity of shed Msb2 was determined using Image J software (bottom). The relative Intensity was calculated by dividing intensity of shed Msb2 under different stress conditions with shed Msb2 under YNB. Control (far left) depicts colony morphology in WT cells that do not carry a tagged version of Msb2.