Amino acid-induced morphogenesis is dependent on amino acid uptake.

Abstract

A. Macrocolonies of wildtype C. albicans (PMRCA18) grown on SXD medium containing 10 mM of the indicated amino acids (X = Pro, Arg, Orn or Asp) and 2% glucose after 48 h of growth at 37 °C (upper panels). Cells scraped from macrocolonies stained with calcofluor white (lower panels); scale bars = 30 μ. B. Amino acid-induced SPS-sensor signaling. Cells expressing Stp2-6XHA (PMRCA48) were grown to log phase in SD medium and induced with 50 μM or 5 mM of the indicated amino acids for 5 min at 30 °C. The levels of latent and processed Stp2 in extracts were analyzed by immunoblotting (upper panels). Similarly, reporter strain (CFG001) carrying an integrated PCAN1-NanoLuc-PEST construct was grown to log phase in SD medium and induced with 50 μM of the indicated amino acids for 2 h at 30 °C. The average luciferase signal (ave. ± CI, 95% CL) are plotted; threshold for significance ≥ 1.5X fold change). C. Macrocolonies of wildtype (WT; PMRCA18) and strains carrying mutations inactivating SPS-sensing pathway components ssy1Δ/Δ (YJA64), ssy5Δ/Δ (YJA53), stp1Δ/Δ (PMRCA59), stp2Δ/Δ (PMRCA57), stp1Δ/Δ stp2Δ/Δ (PMRCA94) and csh3Δ/Δ (PMRCA12) grown on the indicated SXD media. D. Constitutively active Stp2* but not Stp1* bypasses the filamentous growth defect of a ssy1 null mutant in the presence of ornithine. Macrocolonies of WT (PMRCA18), STP1* (PMRCA23), STP2* (PMRCA44), ssy1-/- STP1* (CFG078), and ssy1-/- STP2* (CFG073) grown on SOD with ornithine (O) as sole nitrogen source. Images in C and D were obtained after 24 h of incubation 37 °C.</p