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Copper‐mediated reversal of defective laccase in a Δ vph1 avirulent mutant of Cryptococcus neoformans
Author(s) -
Zhu Xudong,
Gibbons Jack,
Zhang Shirong,
Williamson Peter R.
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03340.x
Subject(s) - laccase , biology , mutant , cryptococcus neoformans , transcription (linguistics) , microbiology and biotechnology , copper , biochemistry , western blot , enzyme , gene , chemistry , linguistics , philosophy , organic chemistry
Summary Previous studies have shown that a Δ vph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper‐containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Δ vph1 mutant as shown by Western blot and immuno‐electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Δ vph1 cells at 30°C by addition of 100 µM copper sulphate. This restoration by copper was found to occur through both transcriptional and post‐translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5′‐untranslated region of CNLAC1 . Copper was also found to restore partial activity to Δ vph1 cells at 0°C, suggesting that cell wall laccase was expressed in the mutant as an apo‐enzyme. Apo‐laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H + )‐ATPase proton pump in copper assembly of laccase in C. neoformans .