J Clin Invest.
VAD1 mutants show reductions in laccase activity and transcription and markedly attenuated virulence in a mouse model. (A) The indicated cells were incubated on malt extract for 5 days at 30°C and examined by India Ink microscopy for analysis of capsule, incubated for 20 minutes on Christensen’s media for analysis of urease activity, and inoculated on asparagine agar containing norepinephrine to assay production of melanin pigment by laccase. Transcription of laccase was measured using an 874–bp fragment of CNLAC1 as previously described (8) from RNA obtained from cells incubated in asparagine media without glucose for 3 hours at 30°C. A PCR-amplified fragment of the ACT1 gene (68) was used as a control. (B) Mice were injected by tail vein (106 of the indicated cells), and progress was followed until they were moribund